Influenza virus-neutralizing antibody and screening method therefor

ABSTRACT

Provided is an anti-influenza virus antibody that exhibits neutralizing activity beyond the barrier of the two groups of influenza viruses categorized according to the conservativeness of hemagglutinin amino acids, a method of producing the same, and a test method for determining whether the subject carries the neutralizing antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/198,147, filed on Aug. 4, 2011, which claims priority to, and thebenefit of, U.S. Applications Nos. 61/380,051, filed Sep. 3, 2010, and61/452,785, filed Mar. 15, 2011. The contents of the aforementionedapplications are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Feb. 5, 2016, is namedSKJ-001DV_Seq_list.txt and is 139,598 bytes in size.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an anti-influenza virus antibody thatexhibits ubiquitous neutralizing activity against all influenza virusesbeyond the barrier of subtypes, a method of producing the same, and amethod of detecting the antibody in a subject.

BACKGROUND OF THE INVENTION

The influenza virus is an RNA envelop virus having a particle size ofabout 100 nm in diameter belonging to the family Orthomyxoviridae. Itoccurs in three types classified according to the antigenicity ofinternal protein thereof: types A, B and C. The influenza virus consistsof an internal nucleocapsid surrounded by a viral envelop having a lipidbilayer structure or a ribonucleic acid (RNA) core associated withnuclear protein and an external glycoprotein. The inner layer of theviral envelop is configured mainly by matrix protein, whereas the outerlayer is mostly configured by a host-derived lipid substance. The RNA ofinfluenza virus assumes a segmentary structure. The influenza thatspreads widely all over the world is caused by type A influenza viruses.Type A viruses have two kinds of envelop glycoproteins, i.e.,hemagglutinin (HA) and neuraminidase (NA). According to antigenicityvariation, HA is classified into 16 subtypes, and NA into 9 subtypes.

In recent years, highly pathogenic H5N1 avian influenza virus has beenrampant worldwide; it could even be said that a new viral strain thatcan be communicated from one person to another could emerge and cause apandemic at any moment. To cope with this situation, a global viraltesting system is being enhanced, and large stockpiling of Tamiflu andthe like as therapeutic drugs, vaccine development, production, andstockpiling are being implemented. However, the situation stands whilemany issues remain to be clarified, including when and how it willemerge, whether Tamiflu and the like will be therapeutically effectivein the event thereof, whether the vaccine developed will be effective,when and to whom it will be inoculated, and, more importantly, when toinstitute a state of high alert, and when to call off it. This isbecause we are going to encounter a situation that has never beenexperienced by human being, where vaccines and therapeutic drugs forpathogens and viruses that have not yet emerged must be stockpiled. Aproblem with vaccine development, in particular, resides in the factthat every year many mutations occur in the hemagglutinin gene on theinfluenza virus genome to cause an antigenic drift (change inantigenicity), which is thought to be the cause of epidemic prevalence.Therefore, inoculating a vaccine that does not match the prevailingsubtype does not have an expected prophylactic effect. The reason why noattempts have been made to develop antibody therapeutic drugs(prophylactic drugs) for influenza virus is that it is feared that amedicine that has been developed with considerable effort is no longeruseful because the virus that should otherwise be neutralized at thetime of development has changed its nature due to an antigenic drift.

The present inventors screened a phage display human antibody librarygenerated from a large number of B lymphocytes collected from oneindividual for twelve influenza virus strains of subtype H3N2 separatedbetween 1968 and 2004, and found that the majority of clones exhibitingneutralizing activity were anti-hemagglutinin antibodies, and that theywere roughly dividable into three groups: those that specificallyneutralize viral strains separated in 1968-1973, viral strains separatedin 1977-1993, and viral strains separated in 1997-2003 (non-patentdocument 1). Although this finding upsets the conventional common notionthat it is meaningless to develop antibody therapeutic drugs(prophylactic drug) for influenza virus, phage antibody libraries havenot been seriously investigated to date since combinations of a heavychain and a light chain do not always reflect the in vitro environmentand for other reasons.

Against this background, three research groups independently succeededin isolating human monoclonal antibodies that neutralize subtype H5influenza viruses (patent documents 1 and 2, non-patent documents 2-4).These antibodies were shown to exhibit neutralizing activity not only onsubtype H5 influenza viruses, but also on other subtypes (e.g., subtypeH1 and the like). However, while the 16 subtypes of hemagglutinin(H1-H16) are classified according to epitope into two major groups(Groups 1 and 2), these antibodies exhibited neutralizing activity onlyagainst Group 1 (e.g., subtypes H1, H2, H5, H6, H8, H9 and the like),and did not exhibit neutralizing activity on subtypes of influenza virusbelonging to Group 2 (e.g., subtypes H3 and H7 and the like). That is,no anti-influenza virus antibody that exhibits a broad range ofneutralizing activity beyond the bather of the two groups based on thesequence of hemagglutinin has been isolated or reported.

X-ray structural analysis has revealed the binding modes of theseantibodies and hemagglutinin, making it evident that the 38-positionamino acid of hemagglutinin has changed to asparagine in subtypes H3 andH7 in Group 2 and undergoes N-type sugar chain modification (non-patentdocuments 4 and 5). Furthermore, it has been reported that introducingan N-type sugar chain modification site into the 38-position of H5caused the binding ability of the neutralizing antibody to decrease by70% (non-patent document 5). It is also known that when an influenzavirus having its hemagglutinin mutated escapes a neutralizing antibody,such mutations accumulate mainly in five regions within thehemagglutinin gene (A, B, C, D and E regions), which reportedlycomprises a neutralizing epitope (non-patent documents 6 and 7). Thesefindings suggest difficulty in acquiring a neutralizing antibody thatacts beyond this barrier between the two groups.

PATENT DOCUMENTS

-   patent document 1: WO 2007/134327-   patent document 2: WO 2008/028946

Non-Patent Documents

-   non-patent document 1: Virology Vol. 397, pp. 322-330, 2010-   non-patent document 2: Proc. Natl. Acad. Sci. USA., Vol. 105, pp.    5986-5991, 2008-   non-patent document 3: PLoS ONE, Vol. 3, pp. 5986-5991, e3942, 2008-   non-patent document 4: Nature Structural & Molecular Biology, Vol.    16, pp. 265-273, 2009-   non-patent document 5: Science, Vol. 324, pp. 246-251, 2009-   non-patent document 6: Nature, Vol. 289, pp. 373-378, 1981-   non-patent document 7: J. Gen. Virol., Vol. 62, pp. 153-169, 1982

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In preparation for a pandemic with subtype H5N1 influenza expected tooccur in the near future and subsequent pandemics with subtype H7 and H9viruses that are likely to occur, there is a high demand for thedevelopment of a prophylactic approach based on a new concept that ismore comprehensive and more reliable than the conventional concept ofinfluenza prophylaxis that vaccines are designed on the basis ofpredicted changes in antigenicity. Accordingly, it is an object of thepresent invention to provide an anti-influenza virus antibody thatexhibits neutralizing activity beyond the barrier between the two groupsbased on the conservativeness of the amino acid sequence ofhemagglutinin protein, desirably an antibody that exhibits neutralizingactivity on all subtypes, i.e., H1 to H16, of influenza virus, and amethod of producing the same. Another object of the present invention isto provide a testing method enabling to determine whether the subjectcarries the above-described universal neutralizing antibody convenientlyand relatively inexpensively.

Means of Solving the Problems

The present inventors collected a large number, 10⁹, of B lymphocytesfrom one individual using apheresis (separated collection of oneparticular component of blood), generated a phage display human antibodylibrary reflecting almost all antibody repertories, and comprehensivelyscreened for antibody clones that bind to inactivated H3N2 influenzavirus by the panning method using the virus as the antigen. Antibodieswere recovered from the selected phage and tested for neutralizingactivities on subtype H3 influenza viruses, which belong to Group 2, andon subtype H1, subtype H2 and subtype H5 influenza viruses, which belongto Group 1. As a result, the inventors surprisingly succeeded inacquiring more than clones of antibodies that exhibit neutralizingactivity not only on subtype H3, which was used as the antigen forscreening, but also on subtype H1, subtype H2 and subtype H5 influenzaviruses, which belong to a group different from the group to which H3subtype belongs. These clones were found to have six differentheavy-chain variable domain (VH) amino acid sequences, all of which weresubjected to IgBLAST search, leading to the judgement that the germlineis VH1-69 for all of them. IgBLAST search revealed that the light-chainvariable domains (VL) present in these six clones were confined to threedifferent germlines.

Since a previously reported neutralizing antibody against subtype H5utilizes VH1-69 or similar VH1-e, and taking into account the bindingmode of the neutralizing antibody and hemagglutinin, revealed by X-raystructural analysis, it is an unexpected finding that the antibodyacquired by the present inventors is capable of neutralizing subtype H3influenza viruses. However, the present inventors idealized that byextensively screening antibodies that react with a certain subtype asthe antigen in the search for an antibody that neutralizes a virus of asubtype in a group different from the group to which the antigenbelongs, an antibody capable of neutralizing all subtypes of influenzavirus beyond the barrier between the groups could be acquired. Hence,the present inventors collected 10⁹ B lymphocytes, a number larger bytwo digits than in conventional cases, from one individual, generated ahuman antibody library that almost completely reflects the donor'santibody repertoire, comprehensively screened for antibody clones thatbind to subtype H3 influenza viruses, and examined their neutralizingactivities on subtype H1, subtype H2 and subtype H5 influenza viruses,thus succeeding for the first time in isolating antibodies capable ofneutralizing influenza viruses of both Group 1 and Group 2.

Analyzing the amino acid sequences of the neutralizing antibodiesobtained revealed that all the neutralizing antibodies utilize theVH1-69 gene as the heavy-chain variable domain V region. Noticeably,clones that exhibited higher neutralizing activity against influenzaviruses of Group 1 than other clones were found to have a deletion ofone amino acid in the heavy-chain variable domain V region. Because theprocess in which antibody-producing cells transform upon growth anddifferentiation stimulation (antibody maturation) occurs as one strandof the DNA double strand encoding the heavy chain and light chainundergoes cleavage, and a mutation is induced in the process ofrepairing the cleavage by DNA polymerase, which frequently causeserrors, the resulting mutations are mostly amino acid substitutionsbased on single-base substitutions. Therefore, the frequency of deletionof one amino acid (3 bases) is extremely low; even if such a deletionoccurs, it mostly has a bad influence, which in turn further reduces theprobability that a mechanism works wherein B cells producing theantibody are stimulated to remain long as memory cells in the body.Taking into account this technical common sense, it is surprising thatthe antibody that neutralized influenza viruses mainly of Group 2 haveacquired rather potent neutralizing activity against influenza virusesbelonging to Group 1 as a result of deletion of one amino acid in aheavy-chain variable domain other than CDR3.

The present inventors conducted further investigations based on thesefindings, and have developed the present invention.

Accordingly, the present invention provides:

-   [1] an isolated antibody that neutralizes both at least one    influenza virus selected from Group 1 consisting of subtype H1,    subtype H2, subtype H5, subtype H6, subtype H8, subtype H9, subtype    H11, subtype H12, subtype H13 and subtype H16 influenza viruses and    at least one influenza virus selected from Group 2 consisting of    subtype H3, subtype H4, subtype H7, subtype H10, subtype H14 and    subtype H15 influenza viruses;-   [2] the antibody according to [1] above, wherein the antibody    neutralizes both at least subtype H1 and/or subtype H5 influenza    virus and subtype H3 influenza virus;-   [3] the antibody according to [1] above, wherein the antibody    neutralizes subtype H1 to subtype H16 influenza viruses;-   [4] the antibody according to [1] above, wherein the heavy-chain    variable domain V region utilizes the VH1-69 or VH1-e gene;-   [5] the antibody according to [4] above, wherein the heavy-chain    variable domain V region has an amino acid deletion;-   [6] the antibody according to [5] above, wherein the heavy-chain    variable domain V region encoded by the VH1-69 or VH1-e gene has at    least a mutation for deleting the 27th glycine;-   [7] the antibody according to [1] above, wherein the light-chain    variable domain V region utilizes the VL1-44, VL1-47 or VL1-51 gene;-   [8] the antibody according to [1] above, wherein the minimum    inhibitory concentration in focus formation inhibition test when the    antibody is converted to type IgG is on the order of 10⁻¹¹ 10⁻¹² M;-   [9] the antibody according to [1] above, wherein the antibody is a    human antibody;-   [10] the antibody according to [1] above, wherein the    complementarity determining region 1 of the heavy-chain variable    domain consists of the amino acid sequence shown by SEQ ID NO:1, and    the complementarity determining region 2 consists of the amino acid    sequence shown by SEQ ID NO:2;-   [11] the antibody according to [10] above, wherein the framework    region 1 of the heavy-chain variable domain consists of the amino    acid sequence shown by SEQ ID NO:3;-   [12] the antibody according to [1] above, wherein the heavy-chain    variable domain V region consists of the amino acid sequence shown    by any one of SEQ ID NOs:4-9;-   [13] the antibody according to [1] above, wherein the heavy-chain    variable domain consists of the amino acid sequence shown by any one    of SEQ ID NOs:10-15;-   [14] the antibody according to [1] above, wherein the heavy-chain    variable domain and the light-chain variable domain consist of the    amino acid sequences shown by one of the following combinations    (a)-(l), respectively:    -   (a) SEQ ID NO:10 and SEQ ID NO:16;    -   (b) SEQ ID NO:10 and SEQ ID NO:17;    -   (c) SEQ ID NO:10 and SEQ ID NO:18;    -   (d) SEQ ID NO:10 and SEQ ID NO:19;    -   (e) SEQ ID NO:10 and SEQ ID NO:20;    -   (f) SEQ ID NO:10 and SEQ ID NO:21;    -   (g) SEQ ID NO:10 and SEQ ID NO:22;    -   (h) SEQ ID NO:11 and SEQ ID NO:23;    -   (i) SEQ ID NO:13 and SEQ ID NO:24;    -   (j) SEQ ID NO:14 and SEQ ID NO:25;    -   (k) SEQ ID NO:15 and SEQ ID NO:26; and    -   (l) SEQ ID NO:12 and SEQ ID NO:70,-   [15] a passive immunotherapeutic agent for influenza comprising the    antibody according to [1] above;-   [16] a method of passive immunotherapy for influenza comprising    administering an effective amount of the antibody according to [1]    above to a mammalian or avian subject that has been infected, or can    get infected, with influenza virus;-   [17] the method according to [16] above, wherein the subject    receiving the administration is a human;-   [18] a method of producing the antibody according to [1] above,    comprising the steps of:    -   (1) providing an antibody library comprising antibody clones        derived from more than about 10⁸ B cells collected from one        individual,    -   (2) contacting an influenza virus of any one of subtypes H1 to        H16 or the hemagglutinin protein of the virus or an        extracellular domain thereof as the antigen with the antibody        library (1), and comprehensively selecting antibody clones that        react with the antigen,    -   (3) recovering an antibody molecule from each antibody clone        selected in the step (2),    -   (4) testing each antibody obtained in the step (3) for        neutralizing activity on both at least one influenza virus        selected from Group 1 and at least one influenza virus selected        from Group 2, and    -   (5) producing an antibody that has neutralized both an influenza        virus belonging to Group 1 and an influenza virus belonging to        Group 2 using a clone that produces the antibody, and recovering        the antibody;-   [19] the method according to [18] above, wherein the antibody is a    human antibody;-   [20] the method according to [18] above, wherein the antibody    library is a phage display library;-   [21] the method according to [20] above, wherein the number of    antibody clones is 10¹⁰ to 10¹¹;-   [22] the method according to [18] above, wherein the B cells are    collected by apheresis;-   [23] the method according to [18] above, wherein the method    comprises using an influenza virus isolate with which the individual    from which the B cells have been collected in the step (2) above has    not been infected or the hemagglutinin protein thereof or an    extracellular domain thereof as the antigen;-   [24] the method according to [23] above, wherein the influenza virus    isolate is of subtype H1, H2 or H3;-   [25] the method according to [23] above, wherein the influenza virus    isolate is of a hemagglutinin subtype with which the individual from    which the B cells have been collected has not been infected;-   [26] the method according to [25] above, wherein the influenza virus    isolate is of subtype H5, H7 or H9;-   [27] the method according to [18] above, comprising testing    neutralizing activity on both at least subtype H1 and/or subtype H5    influenza virus and subtype H3 influenza virus in the step (4)    above;-   [28] the method according to [20] above, further comprising the step    of converting the antibody to type IgG;-   [29] a method of detecting the antibody according to [1] above in a    subject, comprising the steps of:    -   (1) inoculating hemagglutinin of the subtype of any one of        subtypes H1 to H16 to the subject,    -   (2) collecting blood from the subject at the time when        antibody-producing cells have been sufficiently proliferated        after inoculation, and    -   (3) examining the blood for the presence or absence of        antibody-producing cells that present an antibody that binds to        both hemagglutinin of a subtype selected from Group 1 and        hemagglutinin of a subtype selected from Group 2, and that has        the heavy-chain variable domain V region encoded by the VH1-69        or VH1-e gene;-   [30] a method of detecting the antibody according to [1] above in a    subject, comprising the steps of:    -   (1) inoculating hemagglutinin of a subtype selected from Group 1        and hemagglutinin of a subtype selected from Group 2 separately        to the subject,    -   (2) collecting blood from the subject at the time when        antibody-producing cells have been sufficiently proliferated        after inoculation of each hemagglutinin, and    -   (3) examining the blood for the presence or absence of        antibody-producing cells that present an antibody that binds to        hemagglutinin of a subtype selected from a group different from        the group to which the inoculated hemagglutinin belongs, and        that has        -   the heavy-chain variable domain V region encoded by the            VH1-69 or VH1-e gene, and the like.

Effect of the Invention

According to the present invention, a human antibody possessingneutralizing activity against all hemagglutinin subtypes of influenzaviruses can be provided. Passive immunization with the neutralizingantibody enables to effectively prevent or treat influenza even in theevent of an antigenic shift, as well as an antigenic drift. The presentinvention also makes it possible to determine whether the subject hasmemory B cells that produce an antibody that exhibits neutralizingactivity on influenza viruses beyond the barrier of the groups.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1-1 and 1-2 show the results of an ELISA determination of thebinding activities of the antibodies screened for from a phage displayhuman antibody library for all 12 different H3N2 virus strains and oneH1N1 virus strain.

FIG. 2 shows which subtype H3 influenza virus strains were used as theantigens in screening for clones classified under Group.11 and Group 22.

FIG. 3 shows the influenza virus strains used for screening forrespective antibody clones classified under Group.11 and the number ofclones isolated.

FIG. 4 shows the amino acid sequences of the VH and VL of clonesclassified under Group.11, wherein the dot (.) shown in the FR1 regionof FR045-092 indicates a deleted amino acid.

FIG. 5 shows the results of a comparison of the amino acids of theheavy-chain variable domains of clones classified under Group.11 andantibodies reported to exhibit neutralizing activity against both H1strains and H5 strains, wherein the dot (.) shown in the FR1 region ofFR045-092 indicates a deleted amino acid.

FIG. 6 shows the results of an ELISA determination of the bindingactivities of clones classified under Group.11 for H3N2 influenza virusstrains.

FIG. 7 presents Western blot diagrams showing that clones classifiedunder Group.11 recognize the HAs of both H3 and H1 strains.

FIG. 8 shows the results of an investigation of the hemagglutinationinhibiting activities of clones classified under Group.11 againstsubtype H3 and subtype H1 influenza viruses.

FIG. 9 shows the results of an investigation of the focus formationinhibiting activities of clones classified under Group.11 againstsubtype H3 and subtype H1 influenza viruses.

FIG. 10 shows the results of an ELISA determination of whether clonesclassified under Group.11 competitively inhibit the binding activity ofthe mouse monoclonal antibody C179 for subtype H3 influenza virus. ELISAwas performed on the influenza A/USSR New Caledonia strain of C179 inthe presence and absence (No Fab-p3) of Fab-p3 antibodies (F022-360,F026-146, F026-427, F045-092, F005-126), wherein F005-126 served as anegative control that did not react to the influenza A/USSR NewCaledonia strain HA.

FIG. 11 shows the results of an ELISA determination of whether the mousemonoclonal antibody C, 179 competitively inhibits the binding activitiesof clones classified under Group.11 for subtype H3 influenza viruses.ELISA was performed to determine the actions of Fab-p3 antibodies(F022-360, F026-146, F026-427, F045-092) on the influenza A/USSR NewCaledonia strain in the presence (C179 (+)) and absence (C179 (−)) ofC179.

FIG. 12 shows the results of an ELISA determination of whether theantibody clone F005-126 shown in FIG. 2, which possesses a broad rangeof strain specificity for subtype H3 influenza viruses, and an antibodyclone that recognizes both subtype H3 and subtype H1 influenza virusesclassified under Group.11 share an epitope. ELISA was performed todetermine the action of Fab-PP type F005-126 on the influenza A/HongKong Aichi strain in the presence and absence (No Fab-p3) of Fab-p3antibodies (F022-360, F026-146, F026-427, F045-092, F005-126, F019-102),wherein Fab-p3 type F005-126 served as a positive control forcompetitive inhibition, and F019-102 as a negative control that does notreact to the influenza A/Hong Kong Aichi strain.

FIG. 13 shows the results of an FACS analysis of the bindabilities ofclones classified under Group.11 for cells expressing hemagglutininderived from subtype H3 influenza viruses, wherein all grey peaks arefrom negative control F008-038, and the solid-line peaks are from a)F026-427, b) F045-092, and c) F49, respectively.

FIG. 14 shows the results of a measurement of the neutralizingactivities of each IgG antibody against subtype H3, subtype H5, subtypeH2 and subtype H1 influenza viruses, wherein the vertical axis indicatesinfection suppression rates (%), and the lateral axis indicates antibodyconcentrations (μg/mL).

FIGS. 15-1 and 15-2 are graphic representations of the reactivities ofFab clones to the influenza virus A/H3N2 Aichi strains HA0 and HA1,wherein grey peaks indicate the results of Mock-transfection, greenpeaks indicate the results of pDisp-Aic68HA0 transfection, pink peaksindicate the results of pDisp-Aic68HA1 transfection, blue peaks indicatethe results of pDisp-Fuk85HA0 transfection, and orange peaks indicatethe results of pDisp-Fuk85HA1 transfection.

FIG. 16 shows the results of an ELISA determination of whether theantibody F004-104, which recognizes the epitope B on the HA molecule,and antibody clones classified under Group.11 share an epitope. ELISAwas performed to determine the actions of the Fab-p3 type monoclonalantibodies F026-427p3, F045-092p3, F004-104p3 and the mouse-derivedanti-influenza A/H3N2 antibody F49 on the influenza A/H3N2 Panama strainin the presence and absence (No IgG) of IgG antibodies (F026-427IgG,F045-092IgG, F004-104IgG).

FIGS. 17-1 and 17-2 show the amino acid sequences of HA1 and HA2 foreach subtype and strain of influenza virus.

FIG. 18 shows the results of a FACS analysis of the bindabilities ofvarious HA antibodies for cells expressing mutated hemagglutininresulting from replacement of the 136th serine residue of hemagglutininderived from the Aic68 strain influenza virus with threonine or alanine,wherein grey peaks indicate the results of Mock-transfection, greenpeaks indicate the results for the HA of wild type Aic68, blue peaksindicate the results for S136T of HA of Aic68, and pink peaks indicatethe results for S136A of HA of Aic68.

FIG. 19 shows the results of a FACS analysis of the bindability ofF045-092 for cells expressing mutated HA1 resulting from mutualreplacement of the 142nd-146th or 133rd-137th amino acids of HA1 derivedfrom various H3N2 influenza viruses, wherein grey peaks indicate theresults of Mock-transfection, green peaks indicate the reactivity to thewild type, pink peaks indicate the reactivity to the chimera 142A towhich the 142nd-146th amino acid sequence has been transplanted, andblue peaks indicate the reactivity to the chimera 133A to which the133rd-137th amino acid sequence has been transplanted.

FIG. 20 shows the three-dimensional structures of the 91st-260th aminoacid portions of mutated HA1 regions resulting from mutual replacementof the 142nd-146th or 133rd-137th amino acids of the HA1 derived fromvarious H3N2 influenza viruses, wherein the receptor-binding regionappears in orange, the 133rd-137th amino acids of Aic68_Wild in blue,the 142nd-146th amino acids in light blue, the 133rd-137th amino acidsof Wyo03_Wild in red, the 142nd-146th amino acids in pink, the133rd-137th amino acids of Fuk85_Wild in green, and the 142nd-146thamino acids in yellow-green.

FIG. 21 shows the three-dimensional structures of the 91st-260th aminoacid portions of the HA1 region of H3N2 influenza viruses, the sites ofHA1 region recognized by various antibodies, and the names of viralstrains used in determining the sites by the EMAC method, wherein thereceptor-binding region is shown in orange, the sites recognized by therespective anti-HA antibodies in pink, amino acid numbers in thereceptor-binding region in black, the amino acid numbers of antigenrecognition sites in blue, and the amino acids contained as antigenrecognition sites in the receptor-binding region in blue.

FIGS. 22-1, 22-2, 22-3, 22-4, and 22-5 are a graphic representation ofthe results of an experiment of competition between various anti-HAantibodies that bind to the sites A, B, C, D, and E in HA1, and theF045-092 antibody, wherein each left graph was generated with F045-092as the competitor, and each right graph was generated with the cp3 typeof anti-HA antibody as the competitor (+: with cp3 antibody, −: withoutcp3 antibody). The viral strains used are shown in the lower left ofeach graph. No Fab-pp: PBS was used in place of pp type antibody; No Ab:PBS was used in place of all antibodies.

DETAILED DESCRIPTION

Herein, influenza viruses include all currently known subtypes and evensubtypes that will possibly be isolated and identified in the future.Currently known subtypes of influenza viruses include subtypesconsisting of a combination of a type of hemagglutinin selected fromamong H1 to H16 and a type of neuraminidase selected from among N1 toN9.

Influenza viruses are roughly divided into two groups according to thesimilarity of the amino acid sequence of hemagglutinin. Herein, thegroup consisting of subtype H1, subtype H2, subtype H5, subtype H6,subtype H8, subtype H9, subtype H11, subtype H12, subtype H13 andsubtype H16 influenza viruses is referred to as Group 1, and the groupconsisting of subtype H3, subtype H4, subtype H7, subtype H10, subtypeH14 and subtype H15 influenza viruses as Group 2. In selecting acategory in these groups, the subtype of neuraminidase is notconsidered. Novel subtypes that will be isolated and identified in thefuture will be classified under either Group 1 or Group 2 according tothe similarity of the amino acid sequence of hemagglutinin.

The present invention provides an isolated antibody that neutralizesboth at least one influenza virus selected from Group 1 (subtype H1,subtype H2, subtype H5, subtype H6, subtype H8, subtype H9, subtype H11,subtype H12, subtype H13 and subtype H16) and at least one influenzavirus selected from Group 2 (subtype H3, subtype H4, subtype H7, subtypeH10, subtype H14 and subtype H15). Preferably, the neutralizing antibodyof the present invention neutralizes at least subtype H1 and/or subtypeH5 influenza viruses in Group 1, and also neutralizes at least subtypeH3 influenza viruses in Group 2. More preferably, the neutralizingantibody of the present invention further neutralizes subtype H9influenza viruses in Group 1, and also further neutralizes subtype H7influenza viruses in Group 2. The neutralizing antibody of the presentinvention particularly preferably neutralizes all of subtype H1 to H16influenza viruses, most preferably neutralizes even influenza viruses ofa novel hemagglutinin subtype that will be isolated and identified inthe future.

The neutralizing antibody of the present invention can be produced by amethod comprising the steps of:

-   -   (1) providing an antibody library comprising antibody clones        derived from more than about 10⁸ B cells collected from one        individual;    -   (2) contacting an influenza virus of any one of subtypes H1 to        H16 or the hemagglutinin protein of the virus or an        extracellular domain thereof as the antigen with the antibody        library provided in the step (1), and comprehensively selecting        antibody clones that react with the antigen;    -   (3) recovering antibody molecules from each antibody clone        selected in the step (2);    -   (4) testing each antibody obtained in the step (3) for        neutralizing activity against at least one influenza virus        selected from Group 1 and at least one influenza virus selected        from Group 2; and    -   (5) producing an antibody that has neutralized both an influenza        virus belonging to Group 1 and an influenza virus belonging to        Group 2 using a clone that produces the antibody, and recovering        the antibody.

The donor from which B cells as the antibody-producing cells forgenerating the antibody library are collected may be any optionallychosen mammal (e.g., humans, swine, horses and the like) or bird(chicken, ducks and the like) that has ever been infected with influenzavirus; the same animal species as the subject to passively immunize withthe neutralizing antibody of the present invention can be chosen asappropriate, with preference given to a human. In the case of a human,the donor's age, sex, vaccination status (vaccinated or not) and thelike are not limited; however, since the donor desirably has as muchexperience with influenza virus infection as possible, the donor ispreferably 20 years or older, more preferably 30 years or older, stillmore preferably 40 years or older, particularly preferably 50 years orolder, which ages, however, are not to be construed as limiting. Becausean antibody that exhibits neutralizing activity beyond the barrier ofthe groups is capable of neutralizing all viral isolates in all groupsand all subtypes, donors having cells that produce the neutralizingantibody are thought to be unlikely to contract every year's seasonalinfluenza. Therefore, a human having no history of contracting type Ainfluenza during a given period in the past is further desirable.

The amount of blood drawn for collecting B cells to be used to preparean ordinary antibody library is about 20 to 30 mL, the number of B cellscontained in this volume of blood is about 10⁷. All the study groupsthat isolated human neutralizing antibodies against the highlypathogenic H5N1 avian influenza virus generated their antibody libraryof about 10¹⁰ clones by collecting an ordinary amount of blood from aplurality of donors, and combining them, whereas the present inventorsattempted to generate an antibody library of a size that reflects theentire antibody repertory with the aim of exhaustively (comprehensively)acquiring antibodies that bind to a certain subtype of hemagglutinin.Usually, the amount of blood drawn from a human in a single operation islimited to about 200 to 300 mL; therefore, the number of B cellscollectable using this method is at most about 10⁸. With this in mind,the present inventors collected B cells contained in a larger amount ofblood from one individual using apheresis. Preferably, the antibodylibrary used for the purpose of the present invention is built from morethan 10⁹ B cells. For example, collection of about 10⁹ B cells from onehuman individual can be achieved by separating B cells from about 3 L ofblood by apheresis.

The antibody-producing cells for generating the antibody library mayfurther comprise antibody-producing cells derived from anotherindividual, as far as about 10⁸ or more, preferably about 10⁹ or more, Bcells derived from one individual are contained. Specifically, forexample, a number of mononuclear cells equivalent to about 3 L of bloodare recovered by apheresis, after which B cells can be isolated andrecovered by, for example, Ficoll-Paque density gradient centrifugationand the like.

Antibody libraries include, but are not limited to, for example, phagedisplay libraries, libraries obtained by immortalizing B cells using EBvirus, hybridoma libraries obtained by fusing B cells and myeloma cells,and the like. Preferably, a phage display library may be used.

Examples of methods of generating a phage display human antibody libraryas mentioned herein include, but are not limited to, the following.

Although the choice of phage used is not particularly limited, afilamentous phage (Ff bacteriophage) is usually preferably used. Methodsof presenting an extraneous protein onto the phage surface include amethod wherein the extraneous protein is expressed and presented as afusion protein with one of the coat proteins g3p(cp3) and g6p(cp6) tog9p(cp9) on the coat protein, and a commonly used method wherein theextraneous protein is fused to the N-terminal side of cp3 or cp8. Phagedisplay vectors include 1) those that introduce an extraneous gene in afused form into the coat protein gene in the phage genome to allow allcoat protein presented onto the phage surface to be presented as afusion protein with the extraneous protein, as well as 2) those thatinsert a gene that encodes a fusion protein separately from a wild typecoat protein gene to concurrently express the fusion protein and thewild type coat protein, and 3) those that allow Escherichia coli havinga phagemid vector harboring a gene that encodes a fusion protein to beinfected with a helper phage having the wild type coat protein gene andproduce phage particles that concurrently express the fusion protein andthe wild type coat protein. In the case 1), fusion with a largeextraneous protein can result in the loss of the infectivity; therefore,in such cases, a method of type 2) or 3) is used in generating theantibody library.

Specifically, useful vectors include those described by Holt et al.(Curr. Opin. Biotechnol., 11: 445-449, 2000). For example, pCES1 (see J.Biol. Chem., 274: 18218-18230, 1999) is an Fab expression type phagemidvector harboring a DNA that encodes a κL chain constant region placeddownstream of the signal peptide of cp3, a DNA that encodes C_(H3), andthe cp3-encoding sequence placed via a His-tag, a c-myc tag, and anamber stop codon (TAG) downstream of the cp3 signal peptide, under thecontrol of one lactose promoter. The vector presents Fab onto the cp3coat protein when introduced into Escherichia coli having an ambermutation. When it is expressed in the HB2151 strain, which does not havean amber mutation, and the like, however, the strain produces a solubleFab antibody. Useful scFv expression type phagemid vectors include, forexample, pHEN1 (J. Mol. Biol., 222:581-597, 1991) and the like.

Meanwhile, helper phages include, for example, M13-KO7, VCSM13 and thelike.

Other phage display vectors include those designed to join a sequencecomprising a codon that encodes cysteine to each of the 3′ terminus ofthe antibody gene and the 5′ terminus of the coat protein gene toexpress the two genes concurrently and separately (not as a fusionprotein), and to allow the antibody to be presented onto the coatprotein on the phage surface via the S—S bond between the introducedcysteine residues (Morphosis Company's CysDisplay™ technology) and thelike.

Kinds of antibody libraries generated in the present invention includenaive/non-immunized libraries, synthetic libraries, immunized librariesand the like.

A naive/non-immunized library is obtained by acquiring V_(H) and V_(L)genes retained by a normal animal by RT-PCR, and randomly cloning thesame into one of the above-described phage display vectors. Usually,mRNA or the like derived from lymphocytes (preferably peripheral bloodlymphocytes) of peripheral blood, bone marrow, tonsil and the like ofnormal animals is used as the template. A library generated byamplifying only mRNA derived from IgM that has not undergone a classswitch due to antigen sensitization to avoid biases related to the Vgene, such as anamnesis, is especially called a naive library.Representative naive/non-immunized libraries include CAT Company'slibrary (see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14:309-314, 1996), MRC Company's library (see Annu. Rev. Immunol., 12:433-455, 1994), Dyax Company's library (see J. Biol. Chem., 1999(ibid.); Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000) and the like.

A synthetic library is generated by choosing a particular antibody genethat is functional in human B cells, and replacing a portion of the Vgene fragment, for example, a portion of the antigen-binding region ofCDR3 and the like, with a DNA that encodes a random amino acid sequencewith an appropriate length. Synthetic libraries are recognized as beingexcellent in antibody expression efficiency and stability because theycan be built with a combination of V_(H) and V_(L) genes that producescFv and Fab that are functional from the beginning. Representativeexamples include Morphosys Company's HuCAL library (see J. Mol. Biol.,296: 57-86, 2000), BioInvent Company's library (see Nat. Biotechnol.,18: 852, 2000), Crucell Company's library (see Proc. Natl. Acad. Sci.USA, 92: 3938, 1995; J. Immunol. Methods, 272: 219-233, 2003) and thelike. When using a synthetic library, it is desirable to use a VH1-69 orVH1-e gene fragment as the V gene fragment of the heavy-chain variabledomain.

An immunized library is generated by preparing mRNA from lymphocytescollected from a human having an elevated blood antibody titer againstthe target antigen, such as a recipient of vaccination, or fromlymphocytes collected from a human artificially immunized with thetarget antigen by external immunization, and the like, in the samemanner as with the above-described naive/non-immunized library, andamplifying the V_(H) and V_(L) genes by RT-PCR. Because the desiredantibody gene is present in the library already at the beginning, thedesired antibody can be obtained even from a library of relatively smallsize. In the case of humans, however, because an antibody specific forthe subtype of the virus inoculated by vaccination gets amplified,vaccination with an influenza virus of one of the hemagglutinin subtypesH1 to H3, against which many antibodies are estimated to exist in thebody, leads to amplification of antibodies possessing a narrow range ofneutralizing activity such that only a particular isolate in the subtypecan be neutralized; it is feared that the desired neutralizing antibodyis masked. Therefore, in vaccination, it is preferable that a vaccinefor an influenza virus of a subtype with which extensive infection hasnot been reported to date (e.g., in the case of a human, H5, H7, H9 andthe like) be inoculated.

The greater the diversity of the library is, the better; in reality, andtaking into account the number of phages handleable in the subsequentpanning operation (10¹¹-10¹³ phages) and the number of phages needed forclone isolation and propagation in ordinary panning (100 to 1,000phages/clone), however, the size of the library is suitably about 10⁸ to10¹¹ clones. Preferably, the size is 10⁹ and 10⁶ clones for the V_(H)and V_(L) genes, respectively, and the number of Fab or scFv clones is10¹⁰ to 10¹¹ clones.

Methods of generating an antibody library by immortalization using EBvirus include, but are not limited to, for example, the method describedin PLos Medicine 4(5): e178 0928-9936 (2007). The majority of personshave immunity against EB virus because they have ever been infected withthe virus in the context of asymptomatic infection with infectiousmononucleosis; when using an ordinary EB virus, however, virions arealso produced, so that appropriate purification must be performed. It isalso preferable to use a recombinant EB virus retaining the capabilityof immortalizing B lymphocytes, but lacking the capability of virionreplication (e.g., lack of the switching gene for transition from latentinfection state to lytic infection state, and the like) as an EB systemthat can never be contaminated with the virus.

Because marmoset-derived B95-8 cells secrete EB virus, B lymphocytes canbe easily transformed using a culture supernatant thereof. Anantibody-producing B cell line can be obtained by, for example,culturing these cells using a medium supplemented with serum andpenicillin/streptomycin (P/S) (e.g., RPMI1640) or a serum-free mediumsupplemented with a cell proliferation factor, thereafter separating theculture supernatant by filtration or centrifugation and the like,suspending therein antibody-producing B lymphocytes at an appropriateconcentration (e.g., about 10⁷ cells/mL), and incubating the suspensionnormally at 20 to 40° C., preferably at 30 to 37° C., normally for about0.5 to 2 hours. When human antibody-producing cells are provided asmixed lymphocytes, it is preferable to previously remove T lymphocytesby allowing them to form an E rosette with, for example, sheeperythrocytes and the like, to increase transformation frequency, becausethe majority of persons have T lymphocytes that are toxic to cellsinfected with EB virus. It is also possible to select lymphocytesspecific for the target antigen by mixing sheep erythrocytes, previouslycoupled with a soluble antigen, with antibody-producing B lymphocytes,and separating the rosette using a density gradient of Percoll and thelike. Furthermore, because antigen-specific B lymphocytes are capped byadding the antigen in large excess so that they no longer present IgGonto the surface, mixing with sheep erythrocytes previously coupled withan anti-IgG antibody results in the formation of a rosette only byantigen-nonspecific B lymphocytes. Therefore, by collecting a layer ofcells that do not form a rosette from this mixture using a densitygradient of Percoll and the like, it is possible to selectantigen-specific B lymphocytes.

Antibody-secreting cells that have acquired the capability of indefiniteproliferation as a result of the transformation can be back-fused withmouse or human myeloma cells in order to stably sustain theantibody-secreting ability. Examples of the myeloma cells include mousemyeloma cells such as NS-1, P3U1, SP2/0, and AP-1, and human myelomacells such as SKO-007, GM 1500-6TG-2, LICR-LON-HMy2, and UC729-6.

Generation of an antibody library by cell fusion can be achievedaccording to ordinary procedures for hybridoma preparation forgenerating a monoclonal antibody. Specifically, an antibody-producinghybridoma can be prepared by fusing a B cell collected from a donor andone of the above-described myeloma cells.

Fusion operation can be performed according to a known method, forexample, the method of Koehler and Milstein [Nature, vol. 256, p. 495(1975)]. Fusion promoters include polyethylene glycol (PEG), Sendaivirus and the like, with preference given to PEG and the like. Althoughthe molecular weight of PEG is not subject to limitations, PEG1000 toPEG6000, which are of low toxicity and relatively low viscosity, arepreferable. Examples of PEG concentrations include about 10-80%,preferably about 30-50%. Useful solutions for diluting PEG includevarious buffer solutions such as serum-free media (e.g., RPMI1640),complete media comprising about 5-20% serum, phosphate buffered saline(PBS), and Tris buffer. DMSO (e.g., about 10-20%) can also be added asdesired. Examples of the pH of the fusion solution include about 4 to10, preferably about 6 to 8.

The ratio by number of B cells and myeloma cells is normally about 1:1to 20:1; the cell fusion can be efficiently achieved by incubationnormally at 20-40° C., preferably at 30-37° C., normally for 1 to 10minutes.

Hybridoma screening and breeding are normally performed using a mediumusable for animal cells (e.g., RPMI1640) comprising 5-20% FCS or aserum-free medium supplemented with cell proliferation factors, with theaddition of HAT (hypoxanthine, aminopterin, thymidine). Examples of theconcentrations of hypoxanthine, aminopterin and thymidine include about0.1 mM, about 0.4 μM and about 0.016 mM and the like, respectively. Forselecting a human+-+-mouse hybridoma, ouabain resistance can be used.Because human cell lines are more susceptible to ouabain than mouse celllines, it is possible to eliminate unfused human cells by adding ouabainat about 10⁻⁷ to 10⁻³ M to the medium.

In selecting a hybridoma, it is preferable to use feeder cells orculture supernatants of certain cells. As the feeder cells, an allogeniccell species having a lifetime limited so that it dies after helping theemergence of hybridoma, cells capable of producing large amounts of agrowth factor useful for the emergence of hybridoma with theirproliferation potency reduced by radio-irradiation and the like, and thelike are used. For example, mouse feeder cells include splenocytes,macrophages, blood, thymocytes and the like; human feeder cells includeperipheral blood mononuclear cells and the like. Cell culturesupernatants include, for example, primary culture supernatants of theabove-described various cells and culture supernatants of variousestablished cell lines.

The step of selecting an antibody against a target antigen by the phagedisplay method is called panning. To be specific, a phage presenting anantigen-specific antibody is concentrated by repeating about 2 to 4times a series of operations of bringing a carrier having an influenzavirus of any one of subtypes H1 to H16 or the hemagglutinin protein ofthe virus or an extracellular domain thereof, immobilized thereon, and aphage library into contact with each other, washing out the unboundphage, thereafter eluting the bound phage from the carrier, andinfecting the phage to Escherichia coli to proliferate the phage. In thepresent invention, the influenza virus serving as the antigen may havebeen inactivated by formalin treatment. It is preferable that theinfluenza virus isolate used as the antigen be one with which theindividual from which the B cells have been collected has never beeninfected. This is because when using as the antigen a viral isolate withwhich the individual has ever been infected, it is feared that anantibody clone that exhibits neutralizing activity only in a narrowrange becomes dominant and masks the neutralizing antibody desired inthe present invention. Therefore, preferably, an influenza virusbelonging to a hemagglutinin subtype with which the individual fromwhich the B cells have been collected has never been infected, orhemagglutinin thereof, can be used as the antigen. Examples includeinfluenza viruses of subtype H5, subtype H7, and subtype H9.Alternatively, when using as the antigen an influenza virus isolatebelonging to any one of subtypes H1 to H3, it is desirable, for example,to use an isolate of a subtype that once prevailed before the birth ofthe individual from which the B cells have been collected.

The cDNA sequences that encode the hemagglutinins of the varioussubtypes are publicly known; recombinant hemagglutinin of any desiredsubtype can be produced using ordinary gene recombination techniques.Furthermore, the trimeric extracellular domain structure ofhemagglutinin can be generated in accordance with a method described inthe above-described non-patent document 6.

Useful carriers for immobilizing the antigen include various carriersfor use in ordinary antigen-antibody reactions or affinitychromatography, for example, insoluble polysaccharides such as agarose,dextran, and cellulose; synthetic resins such as polystyrene,polyacrylamide, and silicon; microplates, tubes, membranes, columns,beads and the like comprising glass, metal and the like; surface plasmonresonance (SPR) sensor chips, and the like. For the antigenimmobilization, physical adsorption may be used, and a method using achemical bond in use for insolubilizing and immobilizing a protein orenzyme and the like is also acceptable. For example, abiotin-(strept)avidin system and the like are preferably used. Forwashing the unbound phage, a blocking solution such as BSA solution(once or twice), a PBS comprising a surfactant such as Tween (3 to 5times) and the like can be used in sequence. A report is availablementioning that the use of citrate buffer solution (pH 5) and the likeis preferable. For elution of the specific phage, an acid (e.g., 0.1 Mhydrochloric acid and the like) is normally used; cleavage with aspecific protease (e.g., a gene sequence that encodes a trypsin cleavagesite can be introduced into the linkage site between the antibody geneand the coat protein gene; in this case, Escherichia coli infection andproliferation are possible even if all the coat protein is expressed inthe form of a fusion protein because the wild-type coat protein ispresented on the surface of the eluted phage), competitive elution witha soluble antigen, or elution by reduction of the S—S bond (e.g., in theaforementioned CysDisplay™, the antigen-specific phage can be recoveredby dissociating the antibody and the coat protein by using a suitablereducing agent after performing panning) is also possible. When elutionhas been performed with an acid, the eluate is neutralized with Tris andthe like, and the eluted phage is then infected to Escherichia coli,which is then cultured, after which the phage is recovered by aconventional method.

In place of immobilizing the antigen onto a carrier, it is possible toexpress a hemagglutinin trimer on the cell membrane using a yeastdisplay.

After the phage presenting the antigen-specific antibody is concentratedby panning, the phage is infected to Escherichia coli, and the cells areseeded onto a plate and subjected to cell cloning. The phage is againrecovered, and the antigen binding activity is confirmed by an antibodytiter assay (e.g., ELISA, RIA, FIA and the like) or a measurementutilizing fluorescence activated cell sorting (FACS) or surface plasmonresonance (SPR).

The step of infecting the phage antibody clone obtained above toEscherichia coli, and recovering the antibody from the culturesupernatant, can be performed by, for example, infecting the phage to astrain of Escherichia coli that does not have an amber mutation (e.g.,HB2151 strain) to produce and secrete soluble antibody molecules in theperiplasm or the medium, lysing the cell wall with lysozyme and thelike, recovering the extracellular fraction, and purifying the fractionusing as the phage display vector the same purification technique as theabove, when using a vector incorporating an amber stop codon at thelinker site between the antibody gene and the coat protein gene.Provided that a His-tag or c-myc tag has been introduced in advance, theantibody can easily be purified using IMAC, an anti-c-myc antibodycolumn and the like. When cleavage with a specific protease is utilizedin panning, the antibody molecule is separated from the phage surface byallowing the protease to act thereon, so that the desired antibody canbe purified by performing the same purification operation. In thepresent invention, since the neutralizing activity of the antibody ishigher about 100 to 1000 fold when the antibody is a complete antibodyof the IgG type than of the Fab type, the plasmid DNA is recovered fromthe phage clone obtained, a sequence corresponding to the domain thatbinds to the Fc of IgG is added by gene manipulation, Escherichia coliis transformed therewith, and the transformant is cultured, as describedin Examples below. The antibody recovered from the culture supernatantis purified using an IgG Sepharose column and then tested forneutralizing activity.

The desired antibody can also be selected from an antibody-producingcell line obtained by immortalization using EB virus or cell fusion by,for example, reacting the above-described antigen, previously labeledwith a fluorescent substance, with immortalized cells or fusion cells,and then separating the cells that bind to the antigen using afluorescence-activated cell sorter (FACS). In this case, a hybridoma orimmortalized B cells that produce an antibody against the target antigencan be selected directly, so that the labor of cloning can be lessenedsignificantly.

Various methods can be used for cloning a hybridoma that produces amonoclonal antibody against the target antigen. Because aminopterininhibits many cell functions, it is preferable to remove it from themedium as soon as possible. However, human hybridomas are maintainedusing an aminopterin-supplemented medium normally for about 4 to 6 weeksafter fusion. It is desirable that hypoxanthine and thymidine be removedafter 1 week or more has elapsed after removal of aminopterin. When aclone has emerged and its diameter has reached about 1 mm, the antibodycontent in the culture supernatant can be measured.

The amount of antibody can be measured by, for example, a method whereinthe hybridoma culture supernatant is added to a solid phase (e.g.,microplates) with the target antigen or a derivative thereof or apartial peptide thereof adsorbed thereto as it is alone, or along with acarrier, an anti-immunoglobulin (IgG) antibody (an antibody against IgGderived from the same animal species as the animal from which theoriginal antibody-producing cells are derived is used) or Protein A,previously labeled with a radioactive substance (e.g., ¹²⁵I, ¹³¹I, ³H,¹⁴C), an enzyme (e.g., β-galactosidase, β-glucosidase, alkalinephosphatase, peroxidase, malate dehydrogenase), a fluorescent substance(e.g., fluorescamine, fluorescein isothiocyanate), a luminescentsubstance (e.g., luminol, luminol derivatives, luciferin, lucigenin) orthe like, is added, and an antibody against the target antigen bound tothe solid phase is detected; a method wherein the hybridoma culturesupernatant is added to a solid phase with an anti-IgG antibody orProtein A adsorbed thereto, a target antigen labeled with the samelabeling agent as the above or a derivative thereof or a partial peptidethereof is added, and an antibody against the target antigen bound tothe solid phase is detected, and the like.

Although limiting dilution is normally used as the cloning method,cloning using soft agar and cloning using FACS (described above) arealso possible. Cloning by limiting dilution can be performed by, forexample, the following procedures, which, however, are not to beconstrued as limiting.

The amount of antibody is measured as described above, and positivewells are selected. Previously, appropriate feeder cells have beenchosen and added to a 96-well plate. Cells are aspirated fromantibody-positive wells and suspended in a complete medium [e.g.,RMPI1640 supplemented with 10% FCS (fetal calf serum) and P/S] to obtaina density of 30 cells/mL; 0.1 mL (3 cells/well) of this suspension isadded to the 96-well plate with feeder cells added thereto; a portion ofthe remaining cell suspension is diluted to 10 cells/mL and seeded toother wells (1 cell/well) in the same way; the still remaining cellsuspension is diluted to 3 cells/mL and seeded to other wells (0.3cells/well). The cells are cultured for about 2 to 3 weeks until avisible clone appears; the amount of antibody is measured, and positivewells are selected and recloned. In the case of human cells, cloning isrelatively difficult, so that a plate containing 10 cells per well isalso prepared. Although a monoclonal antibody-producing hybridoma can beobtained normally by two times of subcloning, it is desirable to repeatrecloning regularly for several more months to confirm the stabilitythereof.

Hybridomas can be cultured in vitro or in vivo.

Methods of in vitro culture include a method comprising graduallyscaling up the production of a monoclonal antibody-producing hybridomaobtained as described above, from a well plate, while keeping the celldensity at, for example, about 10⁵ to 10⁶ cells/mL, and graduallylowering the FCS concentration.

Methods of in vivo culture include, for example, a method comprising anintraperitoneal injection of mineral oil into a mouse (a mouse that ishistocompatible with the parent strain of the hybridoma) to induceplasmacytoma (MOPC), intraperitoneally injecting about 10⁶ to 10⁷ cellsof the hybridoma 5 to 10 days later, and collecting ascites fluid underanesthesia 2 to 5 weeks later.

Separation and purification of the monoclonal antibody are performedaccording to a method of immunoglobulin separation and purification[e.g., salting-out, alcohol precipitation, isoelectric pointprecipitation, electrophoresis, adsorption-desorption with an ionexchanger (e.g., DEAE, QEAE), ultracentrifugation, gel filtration,specific purification comprising selectively collecting the antibody bymeans of an antigen-coupled solid phase or an active adsorbent such asprotein A or protein G, and dissociating the linkage to obtain theantibody, and the like] in the same manner as with the ordinaryseparation and purification of a polyclonal antibody.

As described above, a monoclonal antibody that binds to an influenzavirus of a particular hemagglutinin subtype can be screened for byculturing the hybridoma in or outside the body of a warm-blooded animal,and harvesting the antibody from a body fluid or culture thereof.

Whether the thus-obtained monoclonal antibody can neutralize influenzavirus beyond the barrier of the groups can be determined by testingneutralizing activities against at least one influenza virus selectedfrom Group 1 and at least one influenza virus selected from Group 2.

Usually, an investigation of neutralizing activity against influenzavirus is often performed by hemagglutination inhibition (HI) test.Influenza virus binds to erythrocytes via the head region ofhemagglutinin, with a sugar chain comprising sialic acid (sialosugarchain) present on the erythrocyte surface as the influenza virusreceptor. As a result, the influenza virus causes the erythrocytes toagglutinate. Because an antibody with neutralizing activity againstinfluenza virus recognizes and binds to hemagglutinin, thehemagglutination property of influenza virus is suppressed by aneutralizing antibody. Therefore, the presence or absence of suppressionof hemagglutination serves as an index of the presence or absence ofneutralizing activity. While the region involved in the hemagglutinationof hemagglutinin is likely to undergo an antigenic drift, the amino acidinvolved in the sialic acid bond in the region tends to be highlyconserved via subtypes of influenza virus, suggesting that theneutralizing antibody of the present invention, which possesses a broadrange of neutralizing activity, recognizes the amino acid as an epitope.Alternatively, neutralizing activity test methods in the presentinvention include, for example, the focus formation inhibition test [J.Clin. Microbiol. Vol. 28, pp. 1308-1313 (1990)]. Specifically, influenzavirus and host cells are contacted with each other in the presence andabsence of the test antibody, and the presence or absence ofneutralizing activity and the level thereof are determined on the basisof whether the test antibody significantly inhibits focus formation dueto viral infection to the host cells.

Although the subtype of the influenza virus to be tested forneutralizing activity is not particularly limited, it is preferable thatat least subtype H1 and/or subtype H5 influenza viruses in Group 1 andat least subtype H3 influenza viruses in Group 2 be included.Alternatively, it is also preferable to further examine neutralizingactivity against influenza virus of subtype H9 in Group 1, and againstinfluenza virus of subtype H7 in Group 2.

Using a clone that produces an antibody molecule confirmed to neutralizeat least one influenza virus selected from Group 1 and at least oneinfluenza virus selected from Group 2 in an antibody moleculeneutralizing activity test, the antibody molecule can be produced inlarge amounts. When the antibody library used is a phage displaylibrary, a phage clone that presents an Fab or scFv of the desiredneutralizing antibody may be infected to Escherichia coli, which may becultured to yield an Fab type antibody or an scFv type antibody, withpreference given to converting them to type IgG antibodies for thepurpose of remarkably enhancing their neutralizing activities. Forexample, conversion of Fab to IgG can be achieved by cutting outfragments that encode VHCH1 and VLCL from the phage DNA, inserting thefragments into a plasmid comprising a fragment that encodes the Fcregion to build a plasmid comprising a DNA that encodes the heavy chainand light chain, transfecting animal cells such as CHO cells therewith,and culturing the cells to allow them to secrete a type IgG antibody inthe culture supernatant. The antibody obtained can be purified andrecovered by a method known per se.

When the antibody library used is a hybridoma prepared by B cellimmortalization using EV virus or cell fusion, it is possible to allowthe hybridoma to produce the antibody molecule in vitro or in vivo asdescribed above, purify the antibody by a conventional method, andrecover the antibody.

For the neutralizing antibody obtained, it is possible to mimic stepsemployed by the immune system (somatic cell mutation and selection) toenhance its affinity for antigens in vitro. Methods of mutagenesis inantibody genes include chain shuffling, random mutagenesis usingEscherichia coli, which is likely to lack its repair system to undergomutations, or error-prone PCR, CDR walking and the like. Selection of aneutralizing antibody with improved affinity for antigens can beachieved by screening for a high-affinity neutralizing antibody from alibrary of mutants generated by the mutagenesis. For example, 1) amethod wherein an antibody phage with high affinity is recovered at lowconcentrations of the antigen used for selection, 2) a method wherein anantibody phage unlikely to leave the antigen is recovered using rigorouswashing conditions, 3) a method wherein an antagonizing reaction isutilized, and the like can be used.

The kinds of the neutralizing antibody of the present invention obtainedas described above mostly utilize the VH1-69 or VH1-e gene as theheavy-chain variable domain V region. This feature is shared by avariety of antibodies that have been reported so far to neutralizeinfluenza viruses of a plurality of subtypes in Group 1; it isinteresting to note that a definite difference exists in the range ofneutralizing activity exhibited, while the same V gene fragment isutilized. A feature of the neutralizing antibody of the presentinvention is that only the VL1-44, VL1-47 or VL1-51 gene is utilized asthe light-chain variable domain V region. Also, the neutralizingantibody of the present invention has a minimum inhibitory concentrationon the order of 10⁻¹¹-10⁻¹² M in a focus formation inhibition test witha type IgG antibody, exhibiting higher levels of neutralizing activitythan those of all antibodies that have been reported so far toneutralize influenza viruses of a plurality of subtypes in Group 1.

An antibody undergoes immunoglobulin gene rearrangements, i.e.,recombination of the V, D, and J regions in the heavy-chain variabledomain or recombination of the V and J regions in the light-chainvariable domain, in the process of B cell differentiation, after whichsomatic cell mutations are induced in the base sequence of the variabledomains. As a result, an antibody having variable domains with higheraffinity for antigens can be produced. Therefore, the kinds of theneutralizing antibody of the present invention, which are B-cell-derivedantibody clones, can even include neutralizing antibodies having anamino acid sequence resulting from a somatic cell mutation in theoriginal immunoglobulin gene. In the formation of an antigen-antibodyconjugate of a neutralizing antibody and an influenza virus antigen, allpoints of contact with the hemagglutinin molecule are present in theheavy-chain variable domain; therefore, the heavy-chain variable domainwas thought to be the site making a substantial contribution to theaffinity of the neutralizing antibody for influenza virus; however,convergence was also observed in the variation of the light-chainvariable domain, which suggested that the light-chain variable domainalso plays a certain role in the neutralizing antibody of the presentinvention. Because the contribution of the complementarity determiningregion 3 (CDR3) to the binding with the antigen is small in theneutralizing antibody of the present invention, the complementaritydetermining regions 1 and 2 present in the heavy-chain variable domain Vregion are more important. Herein, a heavy-chain variable domain Vregion (light-chain variable domain V region) refers to a V region afterrearrangement to constitute the variable domain of the heavy chain(light chain), and can be, for example, a region comprising theframework regions 1, 2 and 3 and the complementarity determining regions1 and 2. A heavy-chain (light-chain) variable domain refers to a portionof antibody that is not the constant region of the Fab region, and canbe, for example, a region comprising the framework regions 1, 2 and 3and the complementarity determining regions 1, 2 and 3. Therefore, theneutralizing antibody of the present invention is preferably, forexample, a neutralizing antibody having the amino acid sequence of SEQID NO:1 as the complementarity determining region 1 of the heavy-chainvariable domain, and also having the amino acid sequence of SEQ ID NO:2as the complementarity determining region 2.

The present inventors also obtained a clone that possesses remarkableneutralizing activity against subtypes H1, H2 and H5, which belong toGroup 1, compared with other clones, while possessing neutralizingactivity against influenza viruses of subtype H3, which belongs to Group2. This clone, unlike other clones, has a structure wherein one aminoacid (the 27th glycine in SEQ ID NO:27) of the framework region 1 in theheavy-chain variable domain is lacked. Therefore, a neutralizingantibody wherein the framework region 1 of the heavy-chain variabledomain consists of the amino acid sequence shown by SEQ ID NO:3 is alsopreferable as the neutralizing antibody of the present invention.

Further specific examples include a neutralizing antibody wherein theheavy-chain variable domain V region consists of the amino acid sequenceshown by any one of SEQ ID NOs:4-9, a neutralizing antibody wherein theheavy-chain variable domain consists of the amino acid sequence shown byany one of SEQ ID NOs:10-15, and a neutralizing antibody wherein theheavy-chain variable domain (SEQ ID NOs:10-15) and the light-chainvariable domain (SEQ ID NOs:16-26 and 70) consist of one of thecombinations of amino acid sequences shown below.

-   -   (a) SEQ ID NO:10, SEQ ID NO:16;    -   (b) SEQ ID NO:10, SEQ ID NO:17;    -   (c) SEQ ID NO:10, SEQ ID NO:18;    -   (d) SEQ ID NO:10, SEQ ID NO:19;    -   (e) SEQ ID NO:10, SEQ ID NO:20;    -   (f) SEQ ID NO:10, SEQ ID NO:21;    -   (g) SEQ ID NO:10, SEQ ID NO:22;    -   (h) SEQ ID NO:11, SEQ ID NO:23;    -   (i) SEQ ID NO:13, SEQ ID NO:24;    -   (j) SEQ ID NO:14, SEQ ID NO:25;    -   (k) SEQ ID NO:15, SEQ ID NO:26: or    -   (l) SEQ ID NO:12, SEQ ID NO:70

Examples also include the base sequences shown by SEQ ID NOs:71-76 asthe base sequences encoding the amino acid sequences of the heavy-chainvariable domains of the foregoing antibodies (SEQ ID NOs:10-15),respectively, and the base sequences shown by SEQ ID NOs:77-88 encodingthe amino acid sequences of the light-chain variable domains (SEQ IDNOs:16-26 and 70), respectively. Therefore, the neutralizing antibody ofthe present invention is exemplified by a neutralizing antibody whereinthe heavy-chain variable domain consists of the amino acid sequenceencoded by the base sequence shown by any one of SEQ ID NOs:71-76, and aneutralizing antibody wherein the heavy-chain variable domain and thelight-chain variable domain consist of the amino acid sequences encodedby one of the combinations of base sequences shown below.

-   -   (a) SEQ ID NO:71, SEQ ID NO:77;    -   (b) SEQ ID NO:71, SEQ ID NO:78;    -   (c) SEQ ID NO:71, SEQ ID NO:79;    -   (d) SEQ ID NO:71, SEQ ID NO:80;    -   (e) SEQ ID NO:71, SEQ ID NO:81;    -   (f) SEQ ID NO:71, SEQ ID NO:82;    -   (g) SEQ ID NO:71, SEQ ID NO:83;    -   (h) SEQ ID NO:72, SEQ ID NO:84;    -   (i) SEQ ID NO:74, SEQ ID NO:85;    -   (j) SEQ ID NO:75, SEQ ID NO:86;    -   (k) SEQ ID NO:76, SEQ ID NO:87; or    -   (l) SEQ ID NO:73, SEQ ID NO:88

These findings demonstrate that the method of the present invention ishighly useful in that it provides not only an antibody capable ofexhibiting neutralizing activity over a broader range than byconventional methods, i.e., beyond the barrier of the groups, but alsoan antibody with higher neutralizing activity than by conventionalmethods.

Because of the broadness of its neutralizing activity, the influenzavirus neutralizing antibody obtained by the method of the presentinvention is thought to recognize a site different from epitopesrecognized by conventional neutralizing antibodies. If the epitoperecognized by the neutralizing antibody of the present invention isclarified, a peptide comprising the amino acid sequence of the epitope(antigenic amino acid sequence) would be useful as a vaccine forinfluenza virus, and a nucleic acid (gene) comprising the base sequencethat encodes the antigenic peptide would be useful as an influenzatesting reagent and testing reagent kit. An immunologically reactiveepitope can be identified using a publicly known method; examplesinclude 1) a method wherein reactivity between a limiting degradationproduct prepared by enzymatically or chemically treating hemagglutininand a neutralizing type IgG antibody acquired in the present inventionis examined, 2) a method wherein the reactivity between an overlappeptide synthesized with reference to an amino acid sequence databaseand a type IgG neutralizing antibody acquired in the present inventionis examined, and the like.

Hemagglutinin undergoes glycosylation as a precursor after transcriptionand translation; glycosylated hemagglutinin is known to be cleaved intothe two subunits HA1 and HA2. Table 1 shows the correspondences betweenthe amino acid sequences and sequence identification numbers of the HA1and HA2 subunits of various influenza viruses.

TABLE 1 HA1 HA2 H3N2 A/Aichi/2/68 SEQ ID NO: 28 SEQ ID NO: 29A/Fukuoka/1/70 SEQ ID NO: 30 SEQ ID NO: 31 A/Tokyo/6/73 SEQ ID NO: 32SEQ ID NO: 33 A/Yamanashi/2/77 SEQ ID NO: 34 SEQ ID NO: 35A/Niigata/102/81 SEQ ID NO: 36 SEQ ID NO: 37 A/Fukuoka/C29/85 SEQ ID NO:38 SEQ ID NO: 39 A/Guizhou/54/89 SEQ ID NO: 40 — A/Kitakyushu/159/93 SEQID NO: 41 — A/Sydney/5/97 SEQ ID NO: 42 SEQ ID NO: 43 A/Panama/2007/99SEQ ID NO: 44 SEQ ID NO: 45 A/Wyoming/3/2003 SEQ ID NO: 46 SEQ ID NO: 47A/New York/55/2004 SEQ ID NO: 48 SEQ ID NO: 49 H3N8 A/WedgeATailed/1977SEQ ID NO: 50 SEQ ID NO: 51 H1N1 A/New Caledonia/20/99 SEQ ID NO: 52 SEQID NO: 53 A/Suita/1/2009 SEQ ID NO: 54 SEQ ID NO: 55 A/Swine/Hokkaido/SEQ ID NO: 56 SEQ ID NO: 57 2/1981 H2N2 A/Japan/305/1957 SEQ ID NO: 58SEQ ID NO: 59 A/Duck/HK/273/1978 SEQ ID NO: 60 SEQ ID NO: 61 H5N2A/Duck/Mongolia/ SEQ ID NO: 62 SEQ ID NO: 63 54/2001 H5N1A/Vietnam/1194/2004 SEQ ID NO: 64 SEQ ID NO: 65 A/Anhui/1/2005 SEQ IDNO: 66 SEQ ID NO: 67 A/Indonesia/5/2005 SEQ ID NO: 68 SEQ ID NO: 69

The antibodies identified by the three groups, reported to be reactiveto subtypes H1 and H5 share the feature of utilizing VH1-69 in theheavy-chain variable domain with the antibody of the present invention;it was suggested that they may share an epitope. Hence, it was predictedthat the epitope is present in the HA2 subunit (a region involved inmembrane fusion). However, unexpectedly, the present inventorsdemonstrated that the neutralizing antibody of the present inventiondoes not compete with an antibody (C179) that competes for an epitopewith the above-described reported antibodies (Nature Structural &Molecular Biology, Vol. 16, pp. 265-273, 2009). This supports the factthat even when utilizing VH1-69 in common, the antibodies do not sharean epitope. Furthermore, the neutralizing antibody of the presentinvention exhibits hemagglutination inhibition (HI) activity, suggestingthat it recognizes and binds to the HA1 subunit (cell receptor-bindingregion).

The isoleucine (the 54th amino acid in the amino acid sequence of SEQ IDNO:27) and phenylalanine (the 55th amino acid in the amino acid sequenceof SEQ ID NO:27) present in the CDR2 region of VH1-69 are continuoushydrophobic amino acid residues known to form a hydrophobic tip andinteract with hydrophobic clusters. The neutralizing antibody of thepresent invention has the aforementioned isoleucine substituted byphenylalanine, and is suggested to have some influence on thebindability to hydrophobic clusters. Hemagglutinin contains ahydrophobic pocket with highly conserved amino acids, i.e., a sialicacid binding site, which site is listed as an epitope candidate (Nature,Vol. 333, pp. 426-431, 1988). Amino acids that form such a sialic acidbinding site include, for example, the 98th tyrosine, 153rd tryptophan,155th threonine, 183rd histidine, 190th glutamic acid, 194th lysine,134th-138th amino acids, and 224th-228th amino acids of A/Aichi/2/68influenza virus HA1 and the like (in the case of an influenza virus of adifferent subtype or strain, corresponding amino acids). Therefore, aregion comprising these amino acids is possibly the epitope. Influenzavirus HA1 has been reported to contain five sites where mutations arelikely to accumulate [A, B (B1, B2), C (C1, C2), D, and E regions] (P.A. Underwood, J. Gen. Virol. vol. 62, 153-169, 1982; Wiley et al.,Nature, vol. 289, 366-378, 1981). In the present invention, region Arefers to the 121st-146th amino acids in SEQ ID NO:28 or a regioncorresponding to the amino acid region; region B1 refers to the155th-163rd amino acids in SEQ ID NO:28 or a region corresponding theamino acid region; region B2 refers to the 155th-163rd amino acids inSEQ ID NO:28 or a region corresponding to the amino acid region; regionC1 refers to the 50th-57th amino acids in SEQ ID NO:28 or a regioncorresponding to the amino acid region; region C2 refers to the275th-279th amino acids in SEQ ID NO:28 or a region corresponding to theamino acid region; region D refers to the 207th-229th amino acids in SEQID NO:28 or a region corresponding to the amino acid region; region Erefers to the 62nd-83rd amino acids in SEQ ID NO:28 or a regioncorresponding to the amino acid region. Because kinds of the antibody ofthe present invention, particularly F045-092, competed with antibodiesthat recognize the vicinities of region A, region B1, and region B2 thatare present in HA1, it is suggested that the vicinities of regions A andB are the epitope.

Because the neutralizing antibody of the present invention is capable ofneutralizing all hemagglutinin subtypes of influenza viruses beyond thebarrier of the groups, it can be an effective prophylactic and/ortherapeutic means not only for seasonal influenza caused by an antigenicdrift, but also for pandemics due to an antigenic shift. Hence, byadministering the neutralizing antibody, passive immunization againstall subtypes of influenza viruses can be performed, which offersexpectations for therapeutic effects on patients who have contractedinfluenza due to any influenza virus, and prophylactic effects onsubjects who are feared to contract, or to be infected, with influenzavirus. Additionally, the neutralizing antibody of the present inventionis thought to be very unlikely to produce adverse reactions because itis an antibody already present in the human body.

The neutralizing antibody of the present invention can be used as apassive immunotherapeutic agent for influenza as it is per se, or afterbeing prepared as a pharmaceutical composition by blending with apharmacologically acceptable carrier.

Here, as the pharmacologically acceptable carrier, various organic orinorganic carrier substances in common use as pharmaceutical materialscan be used, which are formulated as excipients, solvents (dispersingagents), solubilizers, suspending agents, stabilizers, isotonizingagents, buffers, pH regulators, soothing agents and the like.Pharmaceutical additives such as preservatives and antioxidants can alsobe used as necessary.

Examples of suitable excipients include lactose, sucrose, D-mannitol,D-sorbitol, starch, a starch, dextrin, crystalline cellulose,low-substitutional hydroxypropylcellulose, carboxymethylcellulosesodium, gum arabic, pullulan, light silicic anhydride, syntheticaluminum silicate, magnesium metasilicoaluminate and the like.

Examples of suitable solvents include water for injection, physiologicalsaline, Ringer's solution, alcohols, propylene glycol, polyethyleneglycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.

Examples of suitable solubilizers include polyethylene glycol, propyleneglycol, D-mannitol, trehalose, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate, sodium salicylate, sodium acetate and the like.

Examples of suitable suspending agents include surfactants such asstearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride, andglyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol,polyvinylpyrrolidone, carboxymethylcellulose sodium, methylcellulose,hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylcellulose; polysorbates, polyoxyethylene hardened castoroil and the like.

Examples of suitable stabilizers include human serum albumin (HSA),sodium pyrosulfite, Rongalite, sodium hydrogen metasulfite and the like.

Examples of suitable isotonizing agents include sodium chloride,glycerin, D-mannitol, D-sorbitol, glucose and the like.

Examples of suitable buffers include buffer solutions such as ofphosphates, acetates, carbonates and citrates, and the like.

Examples of suitable pH regulators include acids or bases, such ashydrochloric acid and sodium hydroxide.

Examples of suitable soothing agents include benzyl alcohol and thelike.

Examples of suitable preservatives include para-oxybenzoic acid esters,chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid,sorbic acid and the like.

Examples of suitable antioxidants include sulfites, ascorbates and thelike.

Examples of dosage forms for the aforementioned pharmaceuticalcomposition include injectable preparations such as injections (e.g.,subcutaneous injections, intravenous injections, intramuscularinjections, intraperitoneal injections, intra-arterial injections andthe like), drip infusions and the like.

These pharmaceutical compositions can be produced by methods in commonuse in the field of drug formulation technology, for example, methodsdescribed in the Japanese Pharmacopoeia and the like. Specific methodsof preparing pharmaceutical preparations are described in detail below.The antibody content in the pharmaceutical composition varies dependingon the dosage form, dose and the like, and is, for example, about 0.1%to 100% by weight.

For example, an injection is produced by dissolving, suspending oremulsifying the antibody, along with a dispersing agent (e.g.,polysorbate 80, polyoxyethylene hydrogenated castor oil 60, polyethyleneglycol, carboxymethylcellulose, sodium alginate and the like), apreservative (e.g., methylparaben, propylparaben, benzyl alcohol,chlorobutanol, phenol and the like), an isotonizing agent (e.g., sodiumchloride, glycerin, D-mannitol, D-sorbitol, glucose and the like) andthe like, in an aqueous solvent (e.g., distilled water, physiologicalsaline, Ringer's solution and the like) or an oily solvent (e.g.,vegetable oils such as olive oil, sesame oil, cottonseed oil and cornoil, propylene glycol and the like). If desired, additives such as asolubilizer (e.g., sodium salicylate, sodium acetate and the like), astabilizer (e.g., human serum albumin and the like), and a soothingagent (e.g., benzyl alcohol and the like) may be used. The injectionliquid may be subjected to a sterilizing treatment such as filtrationsterilization using a membrane filter and the like as required, and isusually filled in an appropriate container such as an ampoule.

The injection can also be used as a fresh supply obtained by dissolving(dispersing) a powder prepared by treating the above-described liquid byvacuum drying and the like. Examples of methods of vacuum drying includelyophilization and a method using the Speedback Concentrator (SAVANTCompany). When performing lyophilization, it is preferable to lyophilizethe sample, cooled below −10° C., using a flask in the laboratory or atray or vial in industrial settings. When the Speedback Concentrator isused, lyophilization is performed at about 0 to 30° C. under a vacuum ofabout 20 mmHg or less, preferably about 10 mmHg or less. It ispreferable to add a buffering agent such as a phosphate to the liquid tobe dried, to obtain a pH of about 3 to 10. The powder preparationobtained by lyophilization, as a long-stable preparation, can beprepared freshly as an injection by dissolving in water for injection,physiological saline, Ringer's solution and the like, or by dispersingin olive oil, sesame oil, cottonseed oil, corn oil, propylene glycol andthe like before use.

As required, the above-described antibody may be used in combinationwith another therapeutic drug. Examples of therapeutic agents includeTamiflu, Relenza, amantadine and the like.

Alternatively, as required, the above-described antibody may be coupledwith another therapeutic drug. The antibody transports the drug to asite where influenza virus is present or the vicinity thereof, andinhibits the entry of the virus into cells, whereas the drug kills thevirus or treats, mitigates or ameliorates symptoms of influenza.Examples of the drug include all drugs that is, or will be, in use astherapeutic drugs for influenza. Such drugs are, for example, syntheticor naturally occurring, low-molecular-weight or high-molecular-weight,proteinous, non-proteinous, nucleic acidic or nucleotidic substances.Coupling of the antibody and the drug is preferably performed via alinker. The linker is exemplified by one comprising a substituted orunsubstituted aliphatic alkylene chain, and having at both ends thereofa group bindable to a functional group of the antibody or drug, forexample, an N-hydroxysuccinimide group, an ester group, a thiol group,an imidocarbonate group, an aldehyde group or the like (Koutai KogakuNyumon, Chijin Shokan, 1994).

The antibody can also be enclosed in a liposome to facilitate thedelivery of a pharmaceutical into cells as required. Preferableliposomes include positively charged liposomes, positively chargedcholesterols, transmembrane peptide binding liposomes and the like(Mamoru Nakanishi et al., Protein, Nucleic Acid and Enzyme, 44:1590-1596 (1999), Shiroh Futaki, Kagaku To Seibutsu, 43: 649-653 (2005),Clinical Cancer Research 59: 4325-4333 (1999) and the like).

The neutralizing antibody of the present invention is administered vianon-oral routes, for example, intravenous, intraperitoneal,intramuscular, subcutaneous, transdermal administration and the like.

The active ingredient antibody content is exemplified by, but is notlimited to, 100 to 2,500 μg/mL per dose, or 1.0 to 10 mg per kg bodyweight for an adult human patient. Frequency of dosing is, for example,once per 1 to 2 weeks in one to several times of administration or onceper 2 to 3 weeks for about 2 months.

The neutralizing antibody of the present invention can be used not onlyfor prophylaxis and/or treatment of human influenza, but also forprophylaxis and/or treatment of influenza in birds such as chicken andnon-human mammals such as pigs and horses animals by administration tothese animals, whereby the risk of human infection can be reduced inadvance. When the neutralizing antibody of the present invention isapplied to these animals, the same techniques of preparingpharmaceutical preparations as the above can be used.

The neutralizing antibody of the present invention has been isolated byscreening antibodies that are originally present in the human body. Itis reasonable to think that such antibodies are already carried byhumans at some frequencies, rather than occurring extremely rarely.Therefore, individuals capable of producing the neutralizing antibody ofthe present invention are thought to be also resistant to new types ofinfluenza. Meanwhile, individuals lacking the capability of producingsuch antibodies can be said to be threatened by possible infection withnew types of influenza. Provided that whether the neutralizing antibodyis carried can be determined by relatively convenient procedures, it canbe judged that preventive measures by passive immunization are desirablytaken preferentially for non-carriers of the neutralizing antibody.

Accordingly, the present invention also provides a method of detectingthe neutralizing antibody of the present invention in a subject,comprising the steps of:

-   -   (1) inoculating hemagglutinin of a any one of subtypes H1 to H16        to the subject,    -   (2) collecting blood from the subject at the time when        antibody-producing cells have been sufficiently proliferated        after inoculation, and    -   (3) examining the blood for the presence of absence of        antibody-producing cells that present an antibody that binds to        both hemagglutinin of a subtype selected from Group 1 and        hemagglutinin of a subtype selected from Group 2, and that has a        heavy-chain variable domain V region encoded by the VH1-69 or        VH1-e gene, or        a method comprising the steps of:    -   (1) inoculating hemagglutinin of a subtype selected from Group 1        and hemagglutinin of a subtype selected from Group 2 separately        to the subject,    -   (2) collecting blood from the subject at the time when        antibody-producing cells have been sufficiently proliferated        after inoculation of each subtype of hemagglutinin, and    -   (3) examining the blood for the presence or absence of        antibody-producing cells that present an antibody that binds to        hemagglutinin of a subtype selected from a group different from        the group to which the inoculated hemagglutinin belongs, and        that has a heavy-chain variable domain V region encoded by the        VH1-69 or VH1-e gene.

The desired neutralizing antibody cannot be detected in a small amountof blood drawn unless B lymphocytes that produce the desired antibodyare proliferated and concentrated. Hence, the present invention isintended to detect the presence or absence of the neutralizing antibodyof the present invention by induction of hemagglutinin inoculation usingas the indexes the binding activity for hemagglutinin belonging to adifferent group, and the utilization of a V gene fragment shared by themajority of kinds of the neutralizing antibody of the present invention.

The present invention is explained in more detail in the following byreferring to Examples, which are mere exemplifications and do not at alllimit the present invention.

EXAMPLES Blood Sampling

An amount of mononuclear cells equivalent to 3 L of blood was collectedby apheresis from a pediatrician born in 1974. Blood sampling wasperformed in May 2004.

Preparation of Human Phage Antibody Library

A human phage antibody library was prepared by the phage display method.Approximately 10⁹ lymphocytes were recovered from the blood componentobtained by the blood sampling with Ficoll-Paque, and RNAs wereisolated. cDNAs were amplified from the RNAs to construct libraries ofantibody Heavy chains (VHs) and Light chains (VLs), respectively. Theclone numbers of Heavy chains and Light chains were about 10⁹ and 10⁶,respectively. Then, Heavy chains and Light chains were combined toconstruct a human phage antibody library of Fab-cp3 type, which was alibrary including about 10¹⁰ clones.

Influenza Virus Strains Used

The following influenza virus strains were used in this Example. Unlessotherwise noted, abbreviations in the subsequent Examples indicate thefollowing influenza virus strains.

(H3N2 type)

Aic68: A/Aichi/2/68, Fuk70: A/Fukuoka/1/70, Tok73: A/Tokyo/6/73, Yam77:A/Yamanashi/2/77, Nii81: A/Niigata/102/81, Fuk85: A/Fukuoka/C29/85,Gui89: A/Guizhou/54/89, Kit93: A/Kitakyushu/159/93, Syd97:A/Sydney/5/97, Pan99: A/Panama/2007/99, Wyo03: A/Wyoming/3/2003, NY04:A/New York/55/2004

(H1N1 type)

NC99: A/New Caledonia/20/99, S106: A/Solomon Island/3/2006 Screening

Screening was performed by the Panning method. An influenza virus straininactivated by formalin treatment was coated onto an immunotube, andantigen-antibody reaction was subsequently performed between the virusstrain coated in the tube and the phage antibody library. After the tubewas washed with PBS, phages bonded to the antigen were eluted with acid,then immediately neutralized, and recovered. The recovered phages wereinfected to Escherichia coli, recovery rate was calculated, and phageantibodies were prepared. Using the phages, the above-mentionedoperations were repeated. These operations were performed three times,and eluted phages were infected to Escherichia coli, which was thencultured overnight on an LBGA plate to give single colonies. Thesecolonies were isolated to prepare Fab-cp3 antibodies, whose bindingactivity against the virus strain used for the screening was confirmedby the ELISA. Using clones showing the binding activity as positiveclones, further analysis was performed.

Preparation of Fab-Cp3 Type Antibodies

The Escherichia coli infected with the phage obtained in the screeningwas inoculated into YT supplemented with 0.05% glucose, 100 μg/mlampicillin and 1 mM IPTG, and shaking cultured at 30° C. overnight.Culture supernatant containing a Fab-cp3 type antibody secreted by theEscherichia coli was recovered by the centrifugation, and used forexperiments including ELISA, competition ELISA, Western blotting, flowcytometer and the like.

ELISA

A solution of virus strain inactivated by formalin treatment was addedto 96-well Maxisorp, which was coated at 37° C. for 1 hr. The virussolution was removed from the well, and 5% BSA/PBS was added to performblocking for 1 hr. After removing the BSA solution, the Escherichia coliculture supernatant containing a Fab-cp3 type antibody was added andallowed to react for 1 hr. After washed with PBS (PBST) supplementedwith 0.05% Tween 20, a rabbit anti-cp3 antibody was added and allowed toreact for 1 hr. After further washing with PBST, a goat anti-rabbitIgG(H+L)-HRP was added and allowed to react for 1 hr. After washed withPBST, a solution of OPD, which is a substrate of HRP, was added andallowed to react at room temperature, the reaction was then quenched by2N sulfuric acid, after which OD was measured at a wavelength of 492 nM.Unless otherwise stated, all reactions were performed at 37° C.

Sequence Analysis of Isolated Antibody Clones

The base sequences of Heavy chain (VH) and Light chain (VL) of clonespositive to the virus antigen were confirmed by a sequencing reaction.

Grouping of Isolated Clones

After the VH base sequences of clones were confirmed, the sequences wereconverted to amino acid sequences, which were compared among theisolated clones. Focusing on similarity of VH amino acid sequences,especially similarity in CDR3 sequences, the clones were grouped.

Western Blotting

Formalin-treated virus strain was subjected to SDS-PAGE under anonreducing condition to be fractionated, and transferred onto a PVDFmembrane. The PVDF membrane was blocked with PBST supplemented with 2.5%skim milk for 1 hr, then washed with PBST, and subjected to a reactionwith Fab-cp3 type antibody in the culture supernatant for 1 hr. Afterwashed with PBST, the membrane was subjected to a reaction with a rabbitanti-cp3 antibody for 1 hr, further washed with PBST, and then subjectedto a reaction with a goat anti-rabbit IgG(H+L)-HRP for 1 hr. Afterwashed with PBST, the membrane was reacted with ECL solution for 4-5minutes, and bands were detected with a CCD camera. All reactions wereperformed at room temperature.

Preparation of Fab-Pp Type Antibodies

Plasmid DNAs of Fab-cp3 type antibody clones were genetically engineeredto convert into those of Fab-pp type (PP is an Fc-binding domain ofprotein A), with which Escherichia coli was transformed. The Escherichiacoli was inoculated into YT supplemented with 0.05% glucose, 100 μg/mlampicillin and 1 mM IPTG, and shaking cultured at 30° C. overnight.Culture supernatant containing a Fab-pp type antibody secreted by theEscherichia coli was recovered by the centrifugation, and after ammoniumsulfate precipitation, dissolved in PBS, purified with IgG-Sepharosecolumn, and used for experiments including HI activity, virusneutralization activity and the like.

HI Activity Measurement

Purified Fab-pp type antibodies were serially diluted with PBS, eachmixed with a 4 HA unit/well virus solution, and allowed to react at roomtemperature for 1 hr. Red blood cells were added and mixed, and allowedto react at room temperature for 30 min to 1 hr. The results were shownwith the dilution ratio of the antibodies.

Binding Activities of Clones Isolated from N Library Against H3N2Influenza Virus Strains

Binding activities of the antibody clones isolated from the N libraryscreening against any of 12 kinds of H3N2 virus strains and an H1N1virus strain used in the screening are shown as results of ELISA. Theseare experiments for confirming what cross reactivity the clones isolatedin the screening show against the virus strains. The results are shownin FIG. 1.

The isolated clones are classified into groups according to the degreeof binding activity against respective viruses, and group numbers areindicated on the leftmost column. The column “Number isolated” next to“Clone name” on the right is the number of clones showing identical VHamino acid sequence out of clones isolated in the screening, and“Isolated virus strain” further next thereto on the right indicates thescreening with which virus strain the clone was isolated.

Numerical values from ELISA have been processed as follows:

not less than 1: rednot less than 0.5 and less than 1: orangenot less than 0.1 and less than 0.5: yellowless than 0.1: white

The Western Blotting (WB) column on the extreme right shows results onlyon clones for which experiments were performed. Clones for which a bandwas detectable in the position of HA are indicated as “HA”, and clonesfor which a band was detected in other position are indicated as “?”,since what they recognize is currently unknown. Blanks indicate noexperiments performed.

Results of N Library Screening

The number of clones picked up in each screening of H3N2 strain (beforeconfirmation by ELISA), and from among them, the screening with whichvirus strain the clones classified as Group 11 and Group 22 (FIG. 1)were isolated, are shown (FIG. 2).

Group 11 is the group of antibody clones that recognize both of H3 andH1 strains as antigens, and Group 22 is the group of antibody clonesthat recognize any of 12 kinds of H3 strains, but do not recognize H1strains.

Status of Isolation of Clones Belonging to Group 11

Screening virus strains with which respective antibody clones classifiedas Group 11 were isolated, and the isolated number on the screening areshown (FIG. 3).

Based on VH sequence of each clone (VL sequence is not taken intoconsideration), the number of isolated clones having identical VHsequence are shown.

Amino Acid Sequences of Clones Belonging to Group 11

Amino acid sequences of VH and VL of clones classified as Group 11 areshown (FIG. 4).

(1) VH Amino Acid Sequence

The clones belonging to Group 11 were found to have the highest identitywith IGHV1-69*01 according to searches for germline with IgBlast ofNCBI. Therefore, IGHV1-69*01 were judged as the germline.

From the result, comparisons of VH amino acid sequences were performedbetween IGHV1-69*01 and the respective clones. “Identity (%) withGermline FR1-FR3” is calculation of identity with the amino acidsequences of FR1 to FR3 of IGHV1-69*01. Parts with different aminoacids, as a result of comparison with the amino acid sequence ofIGHV1-69*01, are highlighted with a changed color. However, as to CDR3and FR4, amino acids different among each sequence of the clones arehighlighted with changed colors.

(2) VL Amino Acid Sequence

Fifteen clones with the VH amino acid sequence identical with that ofF022-360 have been isolated so far. The VL sequences of 12 clones out ofthese were subjected to sequence analysis, resulting in identificationof 7 kinds of VLs. As a result, it was found that the VH sequencepossessed by F022-360 has 7 kinds of VH-VL combinations. Of the 7 kinds,the VL sequence highlighted with gray is the combination of F022-360.For clones with other VH sequences, the VL sequence of only theindicated clone has been confirmed, and therefore only the one kind ofcombination has been confirmed so far. The VL sequence of F026-245 hasnot been confirmed.

The germline for the amino acid sequence of each VL was determined asone with the highest identity according to searches with IgBlast ofNCBI, like for VHs. Since some, 3 kinds of germlines were detected, eachsequence of the clones was compared against IGLV1-44*01. Different aminoacids are highlighted with a changed color.

Comparison of VH Amino Acid Sequences Between Previously Reported Clonesthat Neutralize H1 and H5 and are Antibodies Whose Germline is 1-69, andClones Belonging to Group 11

Human antibodies that neutralize both of H1 and H5 strains had beenpreviously reported in 3 articles (exactly, 4 articles), the germline ofany of which clones is IGHV1-69. Thus, comparison of amino acidsequences were performed between these clones, and the germlineIGHV1-69*01 or the clones classified as Group 11 (FIG. 5).

Amino acids identical with those in IGHV1-69*01 are shown with bars.

Volumes and pages of each of the articles are as follows:

-   -   2009 Nat. Struct. Mol. Biol.: Vol. 16 265-273    -   2009 Science: Vol. 324 246-251 (2008 PLoS One: Vol. 3 e3942 is        the article related to isolation of the clone.)    -   2008 PNAS: Vol. 105 5986-5991

Binding Activities of Clones Belonging to Group 11 Against H3N2Influenza Virus Strains

The binding activities of the clones belonging to Group 11 against 12kinds of H3 strains and an H1 strain used in the screening wereconfirmed by the ELISA. Assays for each virus strain were performed induplicate, and the mean value and standard deviation were calculated(FIG. 6).

Western Blotting of Clones Belonging to Group 11

These are experiments illustrating that the clones belonging to Group 11recognize HAs of both of H3 and H1 strains (FIG. 7).

All samples were subjected to SDS-PAGE under nonreducing conditions.

Shown in the upper half of FIG. 7 are datas on F022-360 and F045-092,and the datas in the lower half of FIG. 7 are on F026-146 and F026-427.The difference between left and right is of exposure time for datacapture.

F032-093 is a positive control for HA of H3 strain A/kitakyushu/159/93,and F078-155 is a positive control for HA of H1 strain A/NewCaledonia/20/99.

HI Activities of Clones Belonging to Group 11

Confirmation of whether the clones classified as Group 11 have HIactivities was performed (FIG. 8).

The HIU is shown as dilution ratio, wherein the antibodies were dilutedfrom the concentration of 100 μg/ml in 2 fold increments.

Neutralizing Activities by Fab-pp Type Antibodies

Confirmation of whether the clones classified as Group 11 neutralize H3and H1 influenza virus strains (FIG. 9).

The inhibition rate of focus formation on the addition of 250 or 100μg/ml of Fab-pp type antibodies is shown as % Inhibition.

Inhibition of ELISA Activity of C179 by Fab-p3

Mouse monoclonal antibody C179, which neutralizes New Caledonia strainof Sobiet Union type Influenza A, was reacted with an immunoplate towhich a vaccine for the strain had been adsorbed, in the presence orabsence of Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092,F005-126; F005-126 is a negative control unreactive with the HA of NewCaledonia strain of Sobiet Union type Influenza A). Then, theimmunoplate was reacted with an HRP-labeled anti-mouse IgG (manufacturedby MBL), and caused to develop color by OPD to detect C179 bound to thevaccine.

No significant difference was observed in values of ELISA between in thepresence and absence of Fab-p3 antibodies (F022-360, F026-146, F026-427,F045-092, F005-126)(FIG. 10). Therefore, it was found that thereactivity of C179 against the vaccine is not inhibited by F022-360,F026-146, F026-427 or F045-092. This suggests the difference ofrecognition epitope between C179 and F022-360, F026-146, F026-427 orF045-092.

Inhibition of ELISA Activity of Fab-p3 by C179

Fab-p3 antibodies (F022-360, F026-146, F026-427, F045-092, F005-126)were reacted with an immunoplate to which a vaccine for New Caledoniastrain of Sobiet Union type influenza A had been adsorbed, in thepresence or absence of C179. Then, the immunoplate was reacted with arabbit anti-p3 polyclonal antibody, followed by a reaction with anHRP-labeled anti rabbit IgG (manufactured by MBL), and caused to developcolor by OPD to detect Fab-p3 antibodies bound to the vaccine.

No significant difference was observed in values of ELISA between in thepresence and absence of C179 (FIG. 11). Therefore, it was found that thereactivity of F022-360, F026-146, F026-427 or F045-092 against thevaccine is not inhibited by C179. This also suggests the difference ofrecognition epitope between F022-360, F026-146, F026-427 or F045-092 andC179.

Inhibition of ELISA Activity of F005-126 by Fab-p3

Fab-PP type monoclonal antibody F005-126 was reacted with an immunoplateto which a vaccine for Aichi strain of Hong Kong type influenza A hadbeen adsorbed, in the presence or absence of Fab-p3 antibodies(F022-360, F026-146, F026-427, F045-092, F005-126, F019-102; F019-102 isa negative control unreactive with Aichi strain of Hong Kong typeinfluenza A). Then, the immunoplate was reacted with an HRP-labeledrabbit IgG, and caused to develop color by OPD to detect Fab-PP typeF005-126 bound to the vaccine.

F005-126 is an antibody that is not reactive with New Caledonia strainof Sobiet Union type influenza A, but widely reactive with pluralstrains of Hong Kong type influenza A, and neutralizes them. BecauseF022-360, F026-146, F026-427 and F045-092 also are widely reactive withplural strains of Hong Kong type influenza A, possible proximity ofrecognition epitope was conceived. For this reason, competitiveinhibitory experiments with F005-126 were performed. When the reactivityof Fab-PP type F005-126 against a vaccine for Aichi strain of Hong Kongtype influenza A in the presence and absence of Fab-p3 antibodies(F022-360, F026-146, F026-427, F045-092, F019-102), no significantdifference in values of ELISA was observed (FIG. 12). In the presence ofFab-p3 type F005-126 as a positive control, the value of ELISA wassignificantly decreased. Therefore, it was found that the reactivity ofF005-126 against the vaccine is not inhibited by F022-360, F026-146,F026-427 or F045-092. This suggests the difference of recognitionepitope between F005-126 and F022-360, F026-146, F026-427 or F045-092.

Reactivity of F026-427 and F045-092 with HA Expressed on 293T Cells

The HA gene of Yamanashi strain of Hong Kong type influenza A wasinserted into a cloning site of expression vector pNOW to preparepNOW-Yam77HA. pNOW-Yam77HA and lipofectamine LTX were mixed, and addedto 293T cells to perform transfection. After 24 hrs of cultivation,transfected cells were recovered, followed by blocking with 2.5%BSA-PBS-0.05% Na—N₃ at 4° C. for 30 min, and reacted with Fab-p3antibody (F026-427, F045-092, F008-038) at 4° C. for 30 min (F008-038 isa negative control unreactive with the HA of Yamanashi strain). Then,the cells were reacted with a rabbit anti-p3 polyclonal antibody, thenwith an Alexa488-labeled anti-rabbit IgG (manufactured by Pierce) toperform FACS analysis. Similarly, as a positive control, the cells werereacted with Hong Kong type influenza A mouse monoclonal antibody F49,then with an Alexa488-labeled anti-mouse IgG (manufactured by Pierce) toperform FACS analysis.

Compared to the negative control F008-038, which is unreactive with theHA of Yamanashi strain of Hong Kong type influenza A, peak shifts inFACS occurred for F026-427 and F045-092 (FIG. 13). Therefore, F026-427and F045-092 were considered to be antibodies against the HA ofYamanashi strain.

Measurements of Neutralizing Activities of Complete Human IgGs AgainstInfluenza Viruses Samples and Reagents 1. Purified Complete Human IgGAntibodies

-   -   F026-427 (Lot. 100614), F045-092 (Lot.100614)

2. Viruses

-   -   The following virus strains were used.    -   Human H3N2; A/Aichi/2/1968 strain, A/Kitakyusyu/159/1993 strain    -   Avian H3N8; A/Budgerigar/Aichi/1/1977 strain    -   Pandemic H1N1; A/Suita/1/2009 pdm strain    -   Swine H1N1; A/Swine/Hokkaido/2/1981 strain    -   Human H1N1; A/New Caledonia/20/1999 strain    -   Human H2N2; A/Okuda/1957 strain    -   Avian H2N2; A/duck/Hong Kong/273/1978 strain    -   Avian reassortant H5N1;    -   A/duck/Mongolia/54/2001(H5N2) strain        HA×A/duck/Mongolia/47/2001(H7N1)    -   strain NA×A/duck/Hokkaido/49/98(H9N2) strain internal    -   Human H5N1; A/Vietnam/1194/2004 strain    -   Human H5N1; A/Anhui/1/2005 strain    -   Human H5N1; A/Indonesia/5/2005 strain

3. Cells and Media

-   -   MDCK cells were subcultured in 10% FCS-containing MEM, and for        cultivation after neutralization test and virus infection, MEM        containing 0.4% BSA but not containing FCS was used.

4. Reagents for PAP Staining

-   -   Mouse monoclonal antibody to influenza A NP (C43)    -   Rabbit anti serum to mouse IgG (whole molecule) cappel 55436

Goat Anti Serum to Rabbit IgG (Whole Molecule) Cappel 55602

-   -   Rabbit Peroxidase anti Peroxidase (PAP) cappel 55968    -   3,3′-Diaminobenzidin Tetrahydrochroride Sigma D5637    -   Hydrogen peroxide special grade reagent Sigma-Aldrich 13-1910-5

Experimental Method

Human IgG antibodies with the VH and VL amino acid sequences ofrespective clones of F026-427 and F045-092 were prepared, and used forneutralization test. Each purified human IgG antibody solution wasdiluted with 0.4% BSA-containing MEM to 250 μg/mL and 100 μg/mL, andadditionally, serially diluted four fold using the 100 μg/mL solution asstock solution. To each of the diluted antibody solutions obtained, anequal amount of influenza virus solution of each subtype adjusted to 100FFU was added, after which a neutralization reaction was performed at37° C. for 1 hr. MDCK cells preliminarily subcultured in 10%FCS-containing MEM were subjected to a monolayer culture in a 96-wellplate, and after washed with PBS(−), virus was adsorbed thereto at 37°C. for 1 hr using 0.4% BSA-containing MEM supplemented with the reactionsolution after the neutralization reaction at 30 μL/well. Theneutralization solution was removed, and after washed with PBS(−) once,0.4% BSA-containing MEM was added at 50 and 16 hrs of cultivation wasperformed at 37° C. in the presence of CO₂. After the culture solutionwas removed, cells were fixed with 100% ethanol, and dried.Subsequently, infected cells were stained by the enzyme antibodytechnique (PAP method), and the infection inhibitory ratio wascalculated by counting the number of infected cells under themicroscope. The results are shown in FIG. 14.

F026-427 showed neutralization activity against human H3N2 strain, birdH3N8 strain, human H1N1 strain and human H2N2 strain, as well as slightneutralization activity against human H5N1 strain. On the other hand,F045-092 showed neutralization activity against human H3N2 strain, birdH3N8 strain, human H1N1 strain, human and bird H2N2 strain, as well asweak neutralization activity against human and bird H5N1 strains. Fromthe above results, it was suggested that F026-427 and F045-092 shows thereactivity with human-derived strains and bird-derived strains, with afew exceptions, but no reactivity with pig-derived strains.

Reactivity of F026-427 and F045-092 with HA0 and HA1 Expressed on 293TCells

HA0 and HA1 genes of influenza A/H3N2 type Aichi strain were insertedinto a cloning site of expression vector pDisplay to preparepDisp-Aic68HA0 and pDisp-Aic68HA1. In the same manner, HA0 and HA1 genesof influenza A/H3N2 type Fukuoka strain were inserted into a cloningsite of expression vector pDisplay to prepare pDisp-Fuk85HA0 andpDisp-Fuk85HA1. These 4 kinds of plasmids and lipofectamine LTX wereeach mixed, and added to 293T cells to perform transfection. Alsoprepared was a sample in which lipofectamine LTX alone was added to 293Twithout plasmid, for a negative control (Mock-transfection). After 24hrs of cultivation, transfected cells were recovered, and blocked with2.5% BSA-PBS-0.05% Na—N₃ at 4° C. for 30 min, followed by a reaction at4° C. for 30 min with Fab-PP antibody (F026-427PP, F045-092PP),anti-influenza A/H3N2 type antibody F49, or rabbit anti-V5 tag antibody.For positive controls, Fab-PP antibody F003-137PP of anti-influenzaA/H3N2 type Aichi strain was reacted at 4° C. for 30 min with cellstransfected with pDisp-Aic68HA0 and pDisp-Aic68HA1, and cells ofMock-transfection. Likewise, for positive controls, Fab-PP antibodyF019-102PP of influenza A/H3N2 type Fukuoka strain was reacted at 4° C.for 30 min with cells transfected with pDisp-Fuk85HA0 andpDisp-Fuk85HA1, and cells of Mock-transfection. Then, cells reacted withFab-PP antibody were reacted with an Alexa488-labeled anti-human IgG(manufactured by Pierce), cells reacted with F49 were reacted with anAlexa488-labeled anti-mouse IgG (manufactured by Pierce), and cellsreacted with rabbit anti-V5 tag antibody were reacted with anAlexa488-labeled anti-rabbit IgG (manufactured by Pierce), respectively,and FACS analysis was performed.

Compared to the control Mock-transfection cells not expressing HA,F045-092PP reacted to cells expressing HA0 and HA1 of influenza A/H3N2type Aichi strain and HA0 and HA1 of influenza A/H3N2 type Fukuiokastrain, causing a peak shift in FACS (FIG. 15-1). Compared to thecontrol Mock-transfection cells not expressing HA, F026-427PP wasslightly reactive to cells expressing HA0 and HA1 of influenza A/H3N2type Aichi strain, causing a peak shift in FACS (FIG. 15-2). V5 tagantibody, which was an antibody for confirming the expression of HA0 andHA1, was shown to be sufficiently expressed in any of HA0 and HA1expressing cells. Also, F004-137PP and F019-102PP sufficiently reactedto HA0 and HA1, respectively. F49 reacted to HA0, but did not react toHAL

Because F026-427 and F045-092 reacted to HA1, it was considered thatepitopes recognized by these antibodies are present on HA1 molecule, andthat the recognition epitopes thereof are different from those of F49,which has a wide range of strain specificity, as with F026-427 andF045-092. The recognition epitopes of antibodies derived from VH1-69germline with a wide range of strain specificity, which has beenreported in recent years, are mainly in the HA2 region, and has beenconsidered to have neutralization activity by inhibiting the fusionactivity. However, the recognition epitopes of the described F026-427and F045-092 derived from VH1-69 germline are in the HA1 region, and asis clear from FIG. 8, it is considered that they show the neutralizationactivity owing to their HI activities. There is no precedent for anantibody with such the property, and hence, these are antibodies with atotally novel property. Furthermore, as is clear from FIG. 16, therecognition epitope of F026-427 and F045-092 were considered to be inthe vicinity of epitope B.

Inhibition of ELISA Activity by F004-104

Fab-p3 type monoclonal antibodies, F026-427p3, F045-092p3 andF004-104p3, and mouse-derived anti-influenza A/H3N2 type antibody F49were reacted with an immunoplate to which a vaccine for influenza A/H3N2type Panama strain had been adsorbed, in the presence and absence (NoIgG) of IgG antibodies (F026-427IgG, F045-092IgG, F004-104IgG). Then, inorder to detect Fab-p3 antibodies bound to the vaccine, a rabbit-derivedanti-p3 antibody was reacted, followed by a reaction with an HRP-labeledanti-rabbit IgG antibody, and caused to develop color by OPD. Inaddition, for detecting F49, an HRP-labeled anti-rabbit IgG antibody wasreacted, and caused to develop color by OPD. The results are shown inFIG. 16.

Significant inhibitions of the ELISA activities between the same kind ofantibodies were entirely observed. F045-092IgG and F004-104IgG inhibitedthe ELISA activities of any of F026-427p3, F045-092p3 and F004-104p3.Also, F026-427IgG inhibited the ELISA activity of F004-104p3. On theother hand, F026-427IgG did not significantly inhibit F045-092p3, whichwas considered to be stemmed from a much stronger binding activity ofF045-092 as compared to that of F026-427. For F49, no significantinhibition of ELISA activity by the IgG antibodies used was observed.

F004-104 is an antibody whose recognition epitope has been demonstrated,as a result of escape mutant analysis, to be in the vicinity of theamino acid sequence of the 159th and 190th positions on HA1 molecule(FIG. 17-1, FIG. 17-2). The recognition epitopes of F026-427 andF045-092 was considered to be near the recognition epitope of F004-104,as a result of competitive ELISA. Accordingly, the recognition epitopesof F026-427 and F045-092 were assumed to be in the vicinity of the aminoacid sequence of the 159th and 190th positions on HA1 molecule.

Reactivities of F045-092 and F026-427 Against Variant of the 136th AminoAcid

The 136th serine residue of HA of Aic68 strain was replaced withthreonine (variant Aic68S136T) or alanine (variant Aic68S136A) toprepare variant HAs. These variants and wild-type Aic68 strain Aic68 wtwere caused to be expressed on 293T cells, and subjected to a reactionwith F026-427, F045-092, F003-137, F035-015 or F033-038, after which thereactivities were examined by the flow cytometry analysis. The resultsare shown in FIG. 18.

As a result, F035-015 and F033-038 showed no change in the reactivityagainst the two variants. The reactivity of F045-092 against Aic68S136Twas slightly weak than that against Aic68 wt. The reactivity of F045-092against Aic68S136A was even weaker. Because the mutation of serineresidue at 136th position affected the recognition of HA by F045-092, itwas assumed that the 136th residue or an amino acid in the vicinitythereof is the HA recognition epitope of F045-092.

FCM Analyses of F045-092 for Chimeric HA 133A and 142A

The amino acid sequence of 142-146th positions of HA of Wyo03 strain wastransplanted into the amino acid sequence of 142-146th positions of HAof Aic68 strain to prepare a chimera HA (Aic68_142A). Reversely, theamino acid sequence of 142-146th positions of HA of Aic68 strain wastransplanted into the amino acid sequence of 142-146th positions of HAof Wyo03 strain to prepare a chimera HA (Wyo03_142A). On the other hand,the amino acid sequence of 142-146th positions of HA of Aic68 strain wastransplanted into the amino acid sequence of 142-146th positions of HAof Fuk85 strain to prepare a chimera HA (Fuk85_142A). Furthermore, theamino acid sequence of 133-137th positions of HA of Wyo03 strain wastransplanted into the amino acid sequence of 133-137th positions of HAof Fuk85 strain to prepare a chimera HA (Fuk85_133A). These variants andwild-type Aic68 strain HA (Aic68_Wild), wild-type Wyo03 strain HA(Wyo03_Wild), wild-type Fuk85 strain HA (Fuk85_Wild) were caused to beexpressed on 293T cells, followed by reaction with F045-092, after whichthe reactivities were examined by the flow cytometric analysis (EMACmethod [epitope mapping through analysis of chimaeras]; Okada et al,Journal of General Virology, vol. 92, 326-335, 2011). The Results areshown in FIGS. 19 and 20.

F045-092 showed a sufficiently strong reactivity to Aic68_Wild, butslightly reacted with chimera Aic68_142A, into which the amino acidsequence of 142-146th positions of Wyo03 strain had been transplanted.This demonstrated that the amino acid sequence of 142-146th positions ofWyo03 strain inhibits the recognition of HA by F045-092. The amino acidsequence of 142-146th positions of Wyo03 strain has many amino acidresidues with relatively high molecular weights, suggesting thepossibility of causing steric hindrance when the antibody binds to HA.On the other hand, F045-092 reacts very weakly to Fuk85_Wild, butstrongly reacts to chimera Fuk85_133A, into which the amino acidsequence of 133-137th positions of Wyo03 strain HA has beentransplanted. In order to effect the recognition of HA by F045-092, itwas considered that the amino acid sequence of 133-137th positions ispreferably of Wyo03 strain type rather than of Fuk85 strain type.Accordingly, it was assumed that the amino acid sequence of 133-137thpositions are the recognition epitope of F045-092, or it is in thevicinity.

HA1 Antigen Recognition Sites of Anti-HA Antibodies Used in CompetitiveStudies

An X-ray crystal structural analysis file of HA of H3 (1HA0) wasdownloaded from the Protein Data Bank, and the three dimensionalstructure of 91-260th amino acid moiety in the HA1 region wasconstructed with Rasmol 2.7.5 software (FIG. 21). Antigen recognitionsites of each H3N2 antibody for each H3N2 influenza virus werepreliminarily predicted according to the EMAC method. As to F033-038,for example, the A region and the B region were estimated to be antigenrecognition sites for Aic68 strain HA.

Competitive Studies Between Anti-HA Antibodies, which Bind to the SitesA, B, C, D and E of HA1, and F045-092 Antibody

Competitive ELISA was performed by the EMAC method between anti-HAantibodies (F041-342, F041-360, F019-102, F004-111, F033-038, F010-073,F010-014, F004-136, F010-077, F008-055, F008-038, F008-046, F010-032,F035-015, F037-115, F004-104, F003-137), whose recognition sites onantigen had been identified, and F045-092 antibody. The results areshown in FIGS. 22-1, 22-2, 22-3, 22-4, and 22-5.

Fab-pp and Fab-cp3 types of each anti-HA antibody were prepared, and thepp type antibody, as well as the cp3 type antibody as a competitor, wereadded to cause a competition on the antigen, after which the bindingactivity of the pp type antibody against the antigen was measured. To bespecific, H3N2 virus strains inactivated with formalin were coated ontoan immunoplate, which was blocked with 5% BSA. Each 50 μl of an optimalconcentration of the Fab-pp antibody, and 20 μg/ml of cp3 type antibodyof F045-092, or Fab-cp3 type antibody prepared by 20-fold dilution ofEscherichia coli culture supernatant, the mixture was added to theimmunoplate after completion of the blocking, and incubated at 37° C.for 1 hr. After washed with PBST, a rabbit anti-streptavidin-HRPantibody was added in order to detect pp type antibodies bound to theantigen, and further incubation was performed at 37° C. for 1 hr. Afterthe plate was washed, OPD, which is a substrate for HRP, was added andallowed to react for 20 min, then the reaction was quenched by 2Nsulfuric acid, after which the OD of the sample was measured with awavelength of 492 nm.

H3N2 type virus strains used were as follows:

-   -   Aic68: A/Aichi/2/68    -   Yam77: A/Yamanashi/2/77    -   Syd97: A/Sydney/5/97    -   Pan99: A/Panama/2007/99

F045-092 antibody did not compete at all with F041-342 and F041-360,which recognize site C, F019-102, which recognizes site E, or F004-111antibody, which recognizes both of C and E sites, and was considered tonot bind sites C and E. On the other hand, anti-HA antibodies (F033-038,F010-073, F010-014, F004-136, F010-077), which recognize both of sites Aand B, were highly competitive against F045-092. As to an antibody(F035-015), which recognizes only site A around the receptor bindingregion, antibodies (F008-055, F008-038, F008-046, F010-032, F037-115,F004-104), which recognize only site B, and an antibody (F003-137),which recognizes site B2/D, although they competed against F045-092antibody, antibodies other than F008-038 did not provide competitiveresults comparable with antibodies that recognize both of sites A and B.A possibility was suggested that F045-092 antibody recognizes sites Aand B, and a receptor binding region that is between them and has a highpreservation level of amino acids over viral types. This is consistentwith that F045-092 showed HI activity.

INDUSTRIAL APPLICABILITY

According to the present invention, a human antibody that exhibitsneutralizing activity on all subtypes of influenza virus can be screenedfor. The present invention also makes it possible to determine inadvance whether the subject carries an antibody against influenza virus.It is an important task for human being to develop methods forpreventing pandemics with new viral strains, for checking the spread ofinfection if occurring, and for developing a truly effective vaccine togain time to inoculation to many persons. Measures being currently takeninclude implementation of a worldwide virus monitoring system, largestockpiling of therapeutic drugs such as Tamiflu, and development,production, and stockpiling of vaccines, but none can tell in which forma new type of virus will emerge until it emerges actually. As a newpromising solution, the present invention will contribute enormously topublic heath and medicine.

1. A method of passive immunotherapy for influenza comprisingadministering an effective amount of an antibody to a mammalian or aviansubject that has been infected, or can get infected, with influenzavirus, wherein the antibody is an isolated antibody comprising a heavychain variable region comprising SEQ ID NO:15 and a light chain variableregion comprising SEQ ID NO:26, whereby the antibody binds influenzavirus HA.
 2. The method according to claim 2, wherein the subjectreceiving the administration is a human.